Synthetic glucopyranosyl lipid adjuvants

ABSTRACT

Compounds, particularly, glucopyranosyl lipid adjuvant (GLA) compounds, having the following structure (I) are provided: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , L 9 , L 10 , Y 1 , Y 2 , Y 3 , Y 4 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , are as defined herein. Pharmaceutical compositions, vaccine compositions, and related methods for inducing or enhancing immune responses, are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/184,703 filed Jun. 5, 2009, whichis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the field of pharmaceutical and vaccinecompositions. More specifically, embodiments described herein relate topharmaceutical and vaccine compositions, as well as related prophylacticand therapeutic methods, wherein the compositions comprise aglucopyranosyl lipid adjuvant (GLA) as described herein.

Description of the Related Art

The immune system of higher organisms has been characterized asdistinguishing foreign agents (or “non-self”) agents from familiar or“self” components, such that foreign agents elicit immune responseswhile “self” components are ignored or tolerated. Immune responses havetraditionally been characterized as either humoral responses, in whichantibodies specific for antigens are produced by differentiated Blymphocytes known as plasma cells, or cell mediated responses, in whichvarious types of T lymphocytes act to eliminate antigens by a number ofmechanisms. For example, CD4+ helper T cells that are capable ofrecognizing specific antigens may respond by releasing soluble mediatorssuch as cytokines to recruit additional cells of the immune system toparticipate in an immune response. Also, CD8+ cytotoxic T cells that arealso capable of specific antigen recognition may respond by binding toand destroying or damaging an antigen-bearing cell or particle. It isknown in the immunological arts to provide certain vaccines according toa variety of formulations, usually for the purpose of inducing a desiredimmune response in a host.

Several strategies for eliciting specific immune responses through theadministration of a vaccine to a host include immunization withheat-killed or with live, attenuated infectious pathogens such asviruses, bacteria or certain eukaryotic pathogens; immunization with anon-virulent infective agent capable of directing the expression ofgenetic material encoding the antigen(s) to which an immune response isdesired; and immunization with subunit vaccines that contain isolatedimmunogens (such as proteins) from a particular pathogen in order toinduce immunity against the pathogen. (See, e.g., Liu, 1998 NatureMedicine 4(5 suppl.):515.) For certain antigens there may be one or moretypes of desirable immunity for which none of these approaches has beenparticularly effective, including the development of vaccines that areeffective in protecting a host immunologically against humanimmunodeficiency viruses or other infectious pathogens, cancer,autoimmune disease, or other clinical conditions.

It has long been known that enterobacterial lipopolysaccharide (LPS) isa potent stimulator of the immune system, although its use in adjuvantshas been curtailed by its toxic effects. A non-toxic derivative of LPS,monophosphoryl lipid A (MPL), produced by removal of the corecarbohydrate group and the phosphate from the reducing-end glucosamine,has been described by Ribi et al (1986, Immunology andImmunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p407-419).

A further detoxified version of MPL results from the removal of the acylchain from the 3-position of the disaccharide backbone, and is called3-O-deacylated monophosphoryl lipid A (3D-MPL). It can be purified andprepared by the methods taught in GB 2122204B, which reference alsodiscloses the preparation of diphosphoryl lipid A, and 3-O-deacylatedvariants thereof. For example, 30-MPL has been prepared in the form ofan emulsion having a small particle size less than 0.2 μm in diameter,and its method of manufacture is disclosed in WO 94/21292. Aqueousformulations comprising monophosphoryl lipid A and a surfactant havebeen described in WO9843670A2.

Bacterial lipopolysaccharide-derived adjuvants to be formulated inadjuvant combinations may be purified and processed from bacterialsources, or alternatively they may be synthetic. For example, purifiedmonophosphoryl lipid A is described in Ribi et at 1986 (supra), and3-O-deacylated monophosphoryl or diphosphoryl lipid A derived fromSalmonella sp. is described in GB 2220211 and U.S. Pat. No. 4,912,094.3D-MPL and the β(1-6) glucosamine disaccharides as well as otherpurified and synthetic lipopolysaccharides have been described (WO98/01139; U.S. Pat. No. 6,005,099 and EP 0 729 473 B1, Hilgers et al.,1986 Int. Arch. Allergy Immunol., 79(4):392-6; Hilgers et at., 1987,Immunology, 60(1); 141-6; and EP 0 549 074 B1). Combinations of 3D-MPLand saponin adjuvants derived from the bark of Quillaja Saponaria molinahave been described in EP 0 761 231B. WO 95/17210 discloses an adjuvantemulsion system based on squalene, α-tocopherol, and polyoxyethylenesorbitan monooleate (TWEEN™-80), formulated with the immunostimulantQS21, and optionally including 3D-MPL. Despite the accessibility of suchcombinations, the use of adjuvants derived from natural products isaccompanied by high production costs, inconsistency from lot to lot,difficulties associated with large-scale production, and uncertaintywith respect to the presence of impurities in the compositional make-upof any given preparation.

Accordingly, there is a need for improved vaccines, and in particularfor vaccines that beneficially contain high-purity, chemically definedadjuvant components that exhibit lot-to-lot consistency and that can bemanufactured efficiently on an industrial scale without introducingunwanted or structurally undefined contaminants. The present inventionprovides compositions and methods for such vaccines, and offers otherrelated advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention in its several aspects is directed to compounds,compositions and methods that advantageously employ certain syntheticglucopyranosyl lipid adjuvants (GLA) as immunomodulators or adjuvants.Therefore, according to one aspect of the invention described herein,there are provided GLA compounds having a structure according to thefollowing formula (I):

or a pharmaceutically acceptable salt thereof, wherein L₁, L₂, L₃, L₄,L₅, L₆, L₇, L₈, L₉, L₁₀, Y₁, Y₂, Y₃, Y₄, R₁, R₂, R₃, R₄, R₅, R₆, are asdefined herein.

The GLA compounds of the present invention have utility over a broadrange of therapeutic applications where induction of specific ornon-specific immune responses is desired. For example, in certainaspects of the invention, there are provided vaccine compositionscomprising one or more GLA compounds as set forth herein in combinationwith an antigen. Such vaccine compositions may be advantageously used inmethods for stimulating antigen-specific immune responses in subjects inneed thereof. In other aspects of the invention, there are providedpharmaceutical compositions comprising one or more GLA compounds as setforth herein, wherein the compositions are substantially devoid ofantigen. Such pharmaceutical compositions may be advantageously used inmethods for stimulating non-specific immune responses in subjects inneed thereof, for example in the treatment of infection, seasonalrhinitis and the like.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain aspects of this invention, and aretherefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 demonstrates IFN-γ cytokine production induced in vivo followingvaccination of mice with compositions of the invention comprisingantigen and GLA.

FIGS. 2A-2F show antibody responses induced in vivo followingvaccination of mice with compositions of the invention comprisingantigen and GLA.

FIG. 3 shows the NF-kB enhancement observed at different concentrationsof an illustrative GLA compound of the invention (Compound IX).

FIGS. 4A-4D show the induction of immunostimulatory cytokines (MIP-1band TNFa) at different concentrations of an illustrative GLA compound ofthe invention (Compound IX).

DETAILED DESCRIPTION OF THE INVENTION

Monophosphoryl lipid A (MPL) and other related adjuvants are known tomediate their effects, at least in part, by acting as agonists ofToll-like receptors (TLR). The glucopyranosyl lipid adjuvant (GLA)compounds of the present invention were rationally designed based upon3D structural considerations in relation to TLR receptor stimulation.More specifically, according to the present invention, by selectivelydefining the acyl chain lengths of the GLA compounds of the inventionsuch that they achieve a “flat” bottom in the three dimensionalstructure of the compounds, an improved fit may be achieved within thebinding site of a TLR receptor, thereby resulting in enhanced TLRstimulation and enhanced immunostimulatory properties. In addition, thesolubility of the GLA compounds of the invention (e.g., in aqueoussolutions) is advantageously improved due to the shortened acyl chainlengths, thereby facilitating efficient and effective compoundformulation. Furthermore, because the acyl chain lengths are tailored tomake the molecule three dimensionally “flat” along the bottom of themolecule, the compounds can be more effectively incorporated withinvesicles, e.g., for liposomal formulations.

Further still, compounds of the invention provide advantageous profilesof potency relative to toxicity. For example, the compounds of theinvention may be used over a broad and relatively high range of dosagesfor achieving a desired level of activity (e.g., adjuvant activity),while nevertheless remaining substantially non-toxic to human cells andto human patients, as assayed, for example, by the levels of tumornecrosis factor produced from human cells over a range ofconcentrations, which quickly rises and levels off unlike other moretoxic TLR4 agonists such as lipopolysaccharide. This cell based assayshould be predictive of lower inflammatory markers like C-reactiveprotein involved in adverse events in human pharmacology. The favorablepotency vs. toxicity profile for the compounds of the invention may beparticularly important, for example, when administering to childrenwhose tolerance to cytokines may be lower, or when the compounds areused in formulations targeted at a large population where more leveledresponses will translate into more consistent clinical outcomes forpeople with a varied responsiveness to TLR agonism. Similarly,regulatory approval will be simplified since target dosing will be moreforgiving and manufacturing simplified when the range of activepharmaceutical ingredient need not be controlled at as strict atolerance level.

Therefore, the present invention in its many embodiments providescompounds, vaccine compositions, adjuvant compositions, pharmaceuticalcompositions and related formulations and methods that include syntheticGLA compounds as described herein. The GLA compounds represent syntheticimmunomodulators which, advantageously relative to adjuvants of theprior art, and in particular, relative to natural product adjuvants, canbe prepared in substantially homogeneous form. Moreover, the GLAcompounds of the invention can be prepared efficiently and economicallythrough large-scale synthetic chemical manufacturing, unlike naturalproduct-derived adjuvants. Because a synthetic adjuvant that ischemically synthesized from defined starting materials to obtain achemically defined product exhibits qualitative and quantitativebatch-to-batch consistency, the GLA compounds of the invention offerbenefits including improved product quality control.

As described herein, GLA compounds, compositions and methods for theiruse include in some embodiments the use of GLA by itself with apharmaceutically acceptable carrier or excipient for immunologicaladjuvant activity (e.g., non-specific immunostimulatory activity),including “adjuvanting” in which GLA administration to a subject may bewholly independent of, and/or separated temporally and/or spatiallyfrom, administration to the subject of one or more antigens againstwhich elicitation or enhancement of an immune response (e.g., anantigen-specific response) in the subject is desired. Other embodimentsinclude the use of GLA in a vaccine composition that also includes oneor a plurality of antigens to which an immune response elicited orenhanced by such a vaccine is desired.

As described herein, these vaccine compositions may in certain relatedembodiments also include one or more toll-like receptor (TLR) agonistand/or one or a plurality of one or more of a co-adjuvant, animidazoquinoline immune response modifier, and a double stem loop immunemodifier (dSLIM). In other related embodiments, a vaccine composition asprovided herein may comprise GLA and one or more recombinant expressionconstructs each comprising a promoter operably linked to a nucleic acidsequence encoding the antigen against which elicitation or enhancementof an immune response (e.g., an antigen-specific response) in thesubject is desired.

GLA

As noted above, because GLA is a chemically synthesized adjuvant it canbe prepared in substantially homogeneous form, which refers to a GLApreparation that is at least 80%, preferably at least 85%, morepreferably at least 90%, more preferably at least 95% and still morepreferably at least 96%, 97%, 98% or 99% pure with respect to the GLAmolecule.

GLA compounds of the present invention have the following formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

L₁, L₂, L₃, L₄, L₅ and L₆ are the same or different and independently—O—, —NH— or —(CH₂)—;

L₇, L₈, L₉, and L₁₀ are the same or different and independently absentor —C(═O)—;

Y₁ is an acid functional group;

Y₂ and Y₃ are the same or different and independently —OH, —SH, or anacid functional group;

Y₄ is —OH or —SH;

R₁, R₃, R₅ and R₆ are the same or different and independently C₈₋₁₃alkyl; and

R₂ and R₄ are the same or different and independently C₆₋₁₁ alkyl.

As used herein, the above terms have the following meaning:

“Alkyl” means a straight chain or branched, noncyclic or cyclic,unsaturated or saturated aliphatic hydrocarbon containing from 1 to 20carbon atoms, and in certain preferred embodiments containing from 11 to20 carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like,including undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, etc.; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkylsinclude cyclopentenyl and cyclohexenyl, and the like. Cyclic alkyls arealso referred to herein as “homocycles” or “homocyclic rings.”Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively). Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

“C₈₋₁₃alkyl” and “C₆₋₁₁alkyl” mean an alkyl as defined above, containingfrom 8-13 or 6-11 carbon atoms, respectively.

“Acid functional group” means a functional group capable of donating aproton in aqueous media (i.e. a Brønsted-Lowry acid). After donating aproton, the acid functional group becomes a negatively charged species(i.e. the conjugate base of the acid functional group). Examples of acidfunctional groups include, but are not limited to: —OP(═O)(OH)₂(phosphate), —OS(═O)(OH)₂ (sulfate), —OS(OH)₂ (sulfite), —C(═O)OH(carboxylate), —OC(═O)CH(NH₂)CH₂C(═O)OH (aspartate),—OC(═O)CH₂CH₂C(═O)OH (succinate), and —OC(═O)CH₂OP(═O)(OH)₂(carboxymethylphosphate).

In more specific embodiments, the present invention provides GLAcompounds of formula (I), wherein L₅ and L₆ are both —O— and L₇, L₈, L₉,and L₁₀ are each —C(═O)—, and the GLA compounds have the followingformula (II):

In more specific embodiments, the present invention provides GLAcompounds of formula (II), wherein R₁, R₃, R₅ and R₆ are each C_(x)alkyl, where x is constant and is selected from an integer from 8-13,and R₂ and R₄ are both C_(x-2) alkyl, and the GLA compounds have thefollowing formula (III):

In other more specific embodiments, the present invention provides GLAcompounds of formula (III), wherein x is selected from an integer from10-12.

In other more specific embodiments, the present invention provides GLAcompounds of formula (III), wherein x is 11, and the GLA compounds havethe following structure (IV):

In still other specific embodiments, the invention provides GLAcompounds of formula (II), wherein Y₁ is —OP(═O)(OH)₂ and Y₂, Y₃ and Y₄are each —OH, and the GLA compounds have the following formula (V):

In other specific embodiments, the invention provides GLA compounds offormula (II), wherein L₁ and L₃ are both —O— and L₂ and L₄ are both—NH—, and the GLA compounds have the following formula (VI):

In yet more specific embodiments, the invention provides GLA compoundsof formula (II), wherein Y₁ is —OP(O)(OH)₂, Y₂, Y₃ and Y₄ are each —OH,L₁ and L₃ are both —O—, and L₂ and L₄ are both —NH—, and the GLAcompounds have the following formula (VII):

In still other specific embodiments, the present invention provides GLAcompounds of formula (II), wherein Y₁ is —OP(O)(OH)₂, Y₂, Y₃ and Y₄ areeach —OH, L₁ and L₃ are both —O—, L₂ and L₄ are both —NH—, R₁, R₃, R₅and R₆ each are C_(x) alkyl where x is constant and is selected from aninteger from 8-13, and R₂ and R₄ are both C_(x-2) alkyl, and the GLAcompounds have the following formula (VIII):

In a more specific embodiments of formula (VIII), x is 11, and theinvention provides a GLA compound having the following structure (IX):

GLA Compounds

As mentioned above, the present invention provides GLA compounds. TheGLA compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples. In general, the GLA compounds of structure (I) may beprepared by the following Reaction Schemes, wherein all substituents areas defined above unless indicated otherwise.

The sugar backbone of representative GLA compounds can be preparedgenerally according to Reaction Scheme 1, wherein G₁, G₂, G₃, G₄, G₅,G₆, G₇, G₈, G₉, and G₁₀ are either the same or different andindependently an appropriate protecting group or hydrogen. Anappropriate sugar, such as (i), can be purchased or prepared accordingto methods known to those skilled in the art. The functional groups ofsugar (i) can then be fully protected using methods known to thoseskilled in the art to obtain (ii). In this respect, one skilled in theart will recognize that an appropriate orthogonal protecting groupstrategy which allows for selective deprotection of the sugar functionalgroups may be employed. Suitable protecting groups include, but are notlimited to silylethers, benzyl ethers, allyloxycarbonyl, acetals, Fmoc,azide, and the like. Deprotection of G₁ results in free alcohol (iii)which can then be coupled with protected sugar (iv) using appropriatecoupling conditions, for example CCl₃CN/NaH, to obtain the desired sugarbackbone (v).

Representative GLA compound tail pieces, wherein L₅ and L₆ are both —O—and L₇, L₈, L₉, and L¹⁰ are each —C(═O)—, can be prepared generallyaccording to Reaction Scheme 2, wherein G¹¹ represents an appropriateprotecting group. Acid compounds of structure (vi) can be purchased orprepared according to methods known to those skilled in the art.Reaction of (vi) with an appropriate reagent, such as methyl hydrogenmalonate, yields ketoester (vii). Reduction of (vii) yields alcohol(viii). One skilled in the art will recognize that under appropriateconditions the keto group of (vii) may be reduced stereospecifically asexemplified in the Examples. Saponification of (viii) yields acid (ix)which can be subsequently protected to yield (x). Treatment of (x) withacid chloride (xi) yields (xii) which upon deprotection yields (xiii).Compounds (ix) and (xiii) may both be converted to a suitably protectedacid chloride derivative by methods known to those skilled in the artand attached to the GLA compound sugar backbone as shown in ReactionScheme 3 below. Although Reaction Scheme 2 depicts synthesis of a GLAcompound tail piece comprising R₁ and R₂, it should be understood thatother tail pieces comprising other alkyl groups (e.g. R₃, R₄, R₅, andR₆) may also be prepared by an analogous method. Other tail pieces withdifferent L₅, L₆, L₇, L₈, L₉, and L₁₀ groups may also be prepared byanalogous methods.

Representative GLA compounds can be prepared generally according toReaction Scheme 3, wherein G₁₂ and G₁₃ are the same or different andindependently represent an appropriate protecting group. Removal of theG₅ protecting group of (v) followed by reaction with acid chloride (xiv)produces (xv). Similarly, removal of the G₈ protecting group from (xv)followed by reaction with acid chloride (xvi) results in (xvii).Deprotection of (xvii) and reaction with acid chloride (xviii) yields(xix). Removal of G₉ and reaction with (xx) then produces the protectedGLA compound (xxi). Global deprotection of (xxi) results in a compoundof structure (II). Although Reaction Scheme 3 depicts the synthesis of acompound of structure (II), one skilled in the art will recognize thatanalogous methods may be employed to produce any compound of structure(I). In addition, one skilled in the art will also recognize that withselection of the appropriate protecting groups, the final deprotectionresults in the desired compound.

The compounds of the present invention may generally be utilized as thefree base or free acid. Alternatively, the compounds of this inventionmay be used in the form of acid or base addition salts. Acid additionsalts of the free amino compounds of the present invention may beprepared by methods well known in the art, and may be formed fromorganic and inorganic acids. Suitable organic acids include maleic,fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic,propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic,cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids. Suitable inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric, and nitric acids.

Similarly, base addition salts of the acid compounds of the presentinvention may be prepared by methods well known in the art, and may beformed from organic and inorganic bases. Suitable organic bases include,but are not limited to, triethylamine and pyridine. Suitable inorganicbases include, but are not limited to, sodium hydroxide, potassiumhydroxide, sodium carbonate, potassium carbonate, and ammonia. Thus, theterm “pharmaceutically acceptable salt” of structure (I) is intended toencompass any and all acceptable salt forms.

In addition, prodrugs are also included within the context of thisinvention. Prodrugs are any covalently bonded carriers that release acompound of structure (I) in vivo when such prodrug is administered to apatient. Prodrugs are generally prepared by modifying functional groupsin a way such that the modification is cleaved, either by routinemanipulation or in vivo, yielding the parent compound. Prodrugs include,for example, compounds of this invention wherein hydroxy, amine orsulfhydryl groups are bonded to any group that, when administered to apatient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus,representative examples of prodrugs include (but are not limited to)acetate, formate and benzoate derivatives of alcohol and aminefunctional groups of the compounds of structure (I). Further, in thecase of a carboxylic acid (COOH), esters may be employed, such as methylesters, ethyl esters, and the like.

With regard to stereoisomers, the compounds of structure (I) may havechiral centers and may occur as racemates, racemic mixtures and asindividual enantiomers or diastereomers. All such isomeric forms areincluded within the present invention, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds of structure(I) may exist as polymorphs, which are included in the presentinvention. In addition, some of the compounds of structure (I) may alsoform solvates with water or other organic solvents. Such solvates aresimilarly included within the scope of this invention.

Antigen

An antigen, for use in certain embodiments of the herein describedvaccine compositions and methods employing GLA, may be any targetepitope, molecule (including a biomolecule), molecular complex(including molecular complexes that contain biomolecules), subcellularassembly, cell or tissue against which elicitation or enhancement ofimmunoreactivity in a subject is desired. Frequently, the term antigenwill refer to a polypeptide antigen of interest. However, antigen, asused herein, may also refer to a recombinant construct which encodes apolypeptide antigen of interest (e.g, an expression construct). Incertain preferred embodiments the antigen may be, or may be derivedfrom, or may be immunologically cross-reactive with, an infectiouspathogen and/or an epitope, biomolecule, cell or tissue that isassociated with infection, cancer, autoimmune disease, allergy, asthma,or any other condition where stimulation of an antigen-specific immuneresponse would be desirable or beneficial.

Preferably and in certain embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammalianpathogen, which antigen or antigenic composition may include acomposition derived from a virus such as from HIV-1, (such as tat, nef,gp120 or gp160), human herpes viruses, such as gD or derivatives thereofor Immediate Early protein such as ICP27 from HSV1 or HSV2,cytomegalovirus ((esp. Human)(such as gB or derivatives thereof),Rotavirus (including live-attenuated viruses), Epstein Barr virus (suchas gp350 or derivatives thereof), Varicella Zoster Virus (such as gpI,II and IE63), or from a hepatitis virus such as hepatitis B virus (forexample Hepatitis B Surface antigen or a derivative thereof), hepatitisA virus, hepatitis C virus and hepatitis E virus, or from other viralpathogens, such as paramyxoviruses: Respiratory Syncytial virus (such asF and G proteins or derivatives thereof), parainfluenza virus, measlesvirus, mumps virus, human papilloma viruses (for example HPV6, 11, 16,18, etc.), flaviviruses (e.g., Yellow Fever Virus, Dengue Virus,Tick-borne encephalitis virus, Japanese Encephalitis Virus) or Influenzavirus (whole live or inactivated virus, split influenza virus, grown ineggs or MDCK cells, or whole flu virosomes (as described by Gluck,Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof,such as HA, NP, NA, or M proteins, or combinations thereof).

In certain other preferred embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammalianpathogen, which antigen or antigenic composition may include acomposition derived from one or more bacterial pathogens such asNeisseria spp, including N. gonorrhea and N. meningitidis (for examplecapsular polysaccharides and conjugates thereof, transferrin-bindingproteins, lactoferrin binding proteins, PilC, adhesins); S. pyogenes(for example M proteins or fragments thereof, C5A protease, lipoteichoicacids), S. agalactiae, S. mutans; H. ducreyi; Moraxella spp, including Mcatarrhalis, also known as Branhamella catarrhalis (for example high andlow molecular weight adhesins and invasins); Bordetella spp, includingB. pertussis (for example pertactin, pertussis toxin or derivativesthereof, filamentous hemagglutinin, adenylate cyclase, fimbriae), B.parapertussis and B. bronchiseptica; Mycobacterium spp., including M.tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M. bovis, M.leprae, M. avium, M. paratuberculosis, M. smegmatis; Legionella spp,including L. pneumophila; Escherichia spp, including enterotoxic E. coli(for example colonization factors, heat-labile toxin or derivativesthereof, heat-stable toxin or derivatives thereof), enterohemorragic E.coli, enteropathogenic E. coli (for example Shiga toxin-like toxin orderivatives thereof); Vibrio spp, including V. cholera (for examplecholera toxin or derivatives thereof); Shigella spp, including S.sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis; Campylobacter spp, including C. jejuni (for exampletoxins, adhesins and invasins) and E. coli; Salmonella spp, including S.typhi, S. paratyphi, S. choleraesuis, S. enteritidis; Listeria spp.,including L. monocytogenes; Helicobacter spp, including H. pylori (forexample urease, catalase, vacuolating toxin); Pseudomonas spp, includingP. aeruginosa; Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani (for example tetanus toxin and derivative thereof),C. botulinum (for example botulinum toxin and derivative thereof), C.difficile (for example clostridium toxins A or B and derivativesthereof); Bacillus spp., including B. anthracis (for example botulinumtoxin and derivatives thereof); Corynebacterium spp., including C.diphtheriae (for example diphtheria toxin and derivatives thereof);Borrelia spp., including B. burgdorferi (for example OspA, OspC, DbpA,DbpB), B. garinii (for example OspA, OspC, DbpA, DbpB), B. afzelii (forexample OspA, OspC, DbpA, DbpB), B. andersonii (for example OspA, OspC,DbpA, DbpB), B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp. including C. trachomatis (for example MOMP,heparin-binding proteins), C. pneumoniae (for example MOMP,heparin-binding proteins), C. psittaci; Leptospira spp., including L.interrogans; Treponema spp., including T. pallidum (for example the rareouter membrane proteins), T. denticola, T. hyodysenteriae; or otherbacterial pathogens.

In certain other preferred embodiments the vaccine formulations of thepresent invention contain an antigen or antigenic composition capable ofeliciting an immune response against a human or other mammalianpathogen, which antigen or antigenic composition may include acomposition derived from one or more parasites (See, e.g., John, D. T,and Petri, W. A., Markell and Voge's Medical Parasitology—9^(th) Ed.,2006, WB Saunders, Philadelphia; Bowman, D. D., Georgis' Parasitologyfor Veterinarians—8^(th) Ed., 2002, WB Saunders, Philadelphia) such asPlasmodium spp., including P. falciparum; Toxoplasma spp., including T.gondii (for example SAG2, SAG3, Tg34); Entamoeba spp., including E.histolytica; Babesia spp., including B. microti; Trypanosoma spp.,including T. cruzi; Giardia spp., including G. lamblia; Leishmania spp.,including L. major; Pneumocystis spp., including P. carinii; Trichomonasspp., including T. vaginalis; or from a helminth capable of infecting amammal, such as: (i) nematode infections (including, but not limited to,Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura,Necator americanus, Ancylostoma duodenale, Wuchereria bancrofti, Brugiamalayi, Onchocerca volvulus, Dracanculus medinensis, Trichinellaspiralis, and Strongyloides stercoralis); (ii) trematode infections(including, but not limited to, Schistosoma mansoni, Schistosomahaematobium, Schistosoma japonicum, Schistosoma mekongi, Opisthorchissinensis, Paragonimus sp, Fasciola hepatica, Fasciola magna, Fasciolagigantica); and (iii) cestode infections (including, but not limited to,Taenia saginata and Taenia solium). Certain embodiments may thereforecontemplate vaccine compositions that include an antigen derived fromSchisostoma spp., Schistosoma mansonii, Schistosoma haematobium, and/orSchistosoma japonicum, or derived from yeast such as Candida spp.,including C. albicans; Cryptococcus spp., including C. neoformans.

Other preferred specific antigens for M. tuberculosis are for example ThRa12, Tb H9, Tb Ra35, Tb38-1, Erd 14, DPV, MTI, MSL, mTTC2 and hTCC1 (WO99/51748). Proteins for M. tuberculosis also include fusion proteins andvariants thereof where at least two, preferably three polypeptides of M.tuberculosis are fused into a larger protein. Preferred fusions includeRa12-TbH9-Ra35, Erd14-DPV-MTI, DPV-MTI-MSL, Erd14DPV-MTI-MSL-mTCC2,Erd14-DPV-MTI-MSL, DPV-MTI-MSL-mTCC2, TbH9-DPV-MTI (WO 99151748).

Certain preferred antigens for Chlamydia include for example the HighMolecular Weight Protein (HWMP) (WO 99/17741), ORF3 (EP 366 412), CT622,CT610, pmpD, UVEB and putative membrane proteins (Props). OtherChlamydia antigens of the vaccine formulation can be selected from thegroup described in WO 99128475. Preferred bacterial vaccines compriseantigens derived from Streptococcus spp, including S. pneumoniae (forexample capsular polysaccharides and conjugates thereof, PsaA, PspA,PdB, streptolysin, choline-binding proteins) and the protein antigenPneumolysin (Biochem Biophys Acta, 1989, 67, 1007; Rubins et al.,Microbial Pathogenesis, 25, 337-342), and mutant detoxified derivativesthereof (WO 90/06951; WO 99/03884). Other preferred bacterial vaccinescomprise antigens derived from Haemophilus spp., including H. influenzaetype B (for example PRP and conjugates thereof), nontypeable H.influenzae, for example OMP26, high molecular weight adhesins, P5, P6,protein D and lipoprotein D, and fimbrin and fimbrin derived peptides(U.S. Pat. No. 5,843,464) or multiple copy variants or fusion proteinsthereof.

Derivatives of Hepatitis B Surface antigen are well known in the art andinclude, inter alia, those PreS1, Pars2 S antigens set forth describedin European Patent applications EP-A414 374; EP-A-0304 578, and EP198474. In one preferred aspect the vaccine formulation of the inventioncomprises the HIV-1 antigen, gp120, especially when expressed in CHOcells. In a further embodiment, the vaccine formulation of the inventioncomprises gD2t as hereinabove defined.

In a preferred embodiment of the present invention vaccines containingthe claimed adjuvant comprise antigen derived from the Human PapillomaVirus (HPV) considered to be responsible for genital warts (HPV 6 or HPV11 and others), and the HPV viruses responsible for cervical cancer(HPV16, HPV18 and others). Particularly preferred forms of genital wartprophylactic, or therapeutic, vaccine comprise L1 particles orcapsomers, and fusion proteins comprising one or more antigens selectedfrom the HPV 6 and HPV 11 proteins E6, E7, L1, and L2. Certain preferredforms of fusion protein include L2E7 as disclosed in WO 96/26277, andproteinD(1/3)-E7 disclosed in GB 9717953.5 (PCT/EP98/05285). A preferredHPV cervical infection or cancer, prophylaxis or therapeutic vaccine,composition may comprise HPV 16 or 18 antigens. For example, L1 or L2antigen monomers, or L1 or L2 antigens presented together as a viruslike particle (VLP) or the L1 alone protein presented alone in a VLP orcapsomer structure. Such antigens, virus like particles and capsomer areper se known. See for example WO94/00152, WO94/20137, WO94/05792, andWO93/02184.

Additional early proteins may be included alone or as fusion proteinssuch as E7, E2 or preferably F5 for example; particularly preferredembodiments include a VLP comprising L1E7 fusion proteins (WO 96/11272).Particularly preferred HPV 16 antigens comprise the early proteins E6 orF7 in fusion with a protein D carrier to form Protein D-E6 or E7 fusionsfrom HPV 16, or combinations thereof; or combinations of E6 or E7 withL2 (WO 96/26277). Alternatively the HPV 16 or 18 early proteins E6 andE7, may be presented in a single molecule, preferably a Protein D-E6/E7fusion. Such vaccine may optionally contain either or both E6 and E7proteins front HPV 18, preferably in the form of a Protein D-E6 orProtein D-E7 fusion protein or Protein D E6/E7 fusion protein. Thevaccine of the present invention may additionally comprise antigens fromother HPV strains, preferably from strains HPV 31 or 33.

Vaccines of the present invention further comprise antigens derived fromparasites that cause Malaria. For example, preferred antigens fromPlasmodia falciparum include RTS,S and TRAP. RTS is a hybrid proteincomprising substantially all the C-terminal portion of thecircumsporozoite (CS) protein of P. falciparum linked via four aminoacids of the preS2 portion of Hepatitis B surface antigen to the surface(5) antigen of hepatitis B virus. Its full structure is disclosed in theInternational Patent Application No, PCT/EP92/02591, published as WO93/10152 claiming priority from UK patent application No. 9124390.7.When expressed in yeast RTS is produced as a lipoprotein particle, andwhen it is co-expressed with the S antigen from HBV it produces a mixedparticle known as RTS,S.

TRAP antigens are described in the International Patent Application No.PCT/GB89/00895 published as WO 90/01496. A preferred embodiment of thepresent invention is a Malaria vaccine wherein the antigenic preparationcomprises a combination of the RTS,S and TRAP antigens. Other plasmodiaantigens that are likely candidates to be components of a multistageMalaria vaccine are P. faciparum MSP1, AMA1, MSP3, EBA, GLURP, RAP1,RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA, PfEXP1,Pfs25, Pfs28, PFS27125, Pfs16, Pfs48/45, Pfs230 and their analogues inPlasmodium spp.

Accordingly, certain herein disclosed embodiment contemplate an antigenthat is derived from at least one infectious pathogen such as abacterium, a virus or a fungus, including an Actinobacterium such as M.tuberculosis or M. leprae or another mycobacterium; a bacterium such asa member of the genus Salmonella, Neisseria. Borrelia, Chlamydia orBordetella; a virus such as a herpes simplex virus, a humanimmunodeficiency virus (HIV), a feline immunodeficiency virus (FIV),cytomegalovirus, Varicella Zoster Virus, hepatitis virus, Epstein BarrVirus (EBV), respiratory syncytial virus, human papilloma virus (HPV)and a cytomegalovirus; HIV such as HIV-1 or HIV-2; a fungus such asAspergillus, Blastomyces, Coccidioides and Pneumocysti or a yeast,including Candida species such as C. albicans, C. glabrata, C. krusei,C. lusitaniae, C. tropicalis and C. parapsilosis; a parasite such as aprotozoan, for example, a Plasmodium species including P. falciparum, P.vivax, P. malariae and P. ovale; or another parasite such as one or moreof Acanthamoeba, Entamoeba histolytica, Angiostrongylus, Schistosomamansonii, Schistosoma haematobium, Schistosoma japonicum,Cryptosporidium, Ancylostoma, Entamoeba histolytica, Entamoeba coli,Entamoeba dispar, Entamoeba hartmanni, Entamoeba polecki, Wuchereriabancrofti, Giardia, and Leishmania.

For example, in GLA-containing vaccine embodiments containing antigensderived from Borrelia sp., the antigens may include nucleic acid,pathogen derived antigen or antigenic preparations, recombinantlyproduced protein or peptides, and chimeric fusion proteins. One suchantigen is OspA. The OspA may be a full mature protein in a lipidatedform by virtue of its biosynthesis in a host cell (Lipo-OspA) or mayalternatively be a non-lipidated derivative. Such non-lipidatedderivatives include the non-lipidated NS1-OspA fusion protein which hasthe first 81 N-terminal amino acids of the non-structural protein (NS1)of the influenza virus, and the complete OspA protein, and another,MDP-OspA is a non-lipidated form of OspA carrying 3 additionalN-terminal amino acids.

Compositions and methods are known in the art for identifying subjectshaving, or suspected of being at risk for having, an infection with aninfectious pathogen as described herein.

For example, the bacterium Mycobacterium tuberculosis cases tuberculosis(TB). The bacteria usually attack the lungs but can also attack thekidney, spine, and brain. If not treated properly, TB disease can befatal. The disease is spread from one person to another in the air whenan infected person sneezes or coughs. In 2003, more than 14,000 cases ofTB were reported in the United States.

Although tuberculosis can generally be controlled using extendedantibiotic therapy, such treatment is not sufficient to prevent thespread of the disease and concerns exist regarding the potentialselection for antibiotic-resistant strains. Infected individuals may beasymptomatic, but contagious, for some time. In addition, althoughcompliance with the treatment regimen is critical, patient behavior isdifficult to monitor. Some patients do not complete the course oftreatment, which can lead to ineffective treatment and the developmentof drug resistance. (e.g., U.S. Pat. No. 7,087,713)

Currently, vaccination with live bacteria is the most efficient methodfor inducing protective immunity against tuberculosis. The most commonMycobacterium employed for this purpose is Bacillus Calmette-Guerin(BCG), an avirulent strain of Mycobacterium Bovis. However, the safetyand efficacy of BCG is a source of controversy and some countries, suchas the United States, do not vaccinate the general public. Diagnosis iscommonly achieved using a skin test, which involves intradermal exposureto tuberculin PPD (protein-purified derivative). Antigen-specific T cellresponses result in measurable induration at the injection site by 48 72hours after injection, which indicates exposure to Mycobacterialantigens. Sensitivity and specificity have, however, been a problem withthis test, and individuals vaccinated with BCG cannot be distinguishedfrom infected individuals. (e.g., U.S. Pat. No. 7,087,713)

While macrophages have been shown to act as the principal effectors ofM. tuberculosis immunity, T cells are the predominant inducers of suchimmunity. The essential role of T cells in protection against M.tuberculosis infection is illustrated by the frequent occurrence of M.tuberculosis in AIDS patients, due to the depletion of CD4 T cellsassociated with human immunodeficiency virus (HIV) infection.Mycobacterium-reactive CD4 T cells have been shown to be potentproducers of gamma-interferon (IFN-gamma), which, in turn, has beenshown to trigger the anti-mycobacterial effects of macrophages in mice.While the role of IFN-gamma in humans is less clear, studies have shownthat 1,25-dihydroxy-vitamin D3, either alone or in combination withIFN-gamma or tumor necrosis factor-alpha, activates human macrophages toinhibit M. tuberculosis infection. Furthermore, it is known thatIFN-gamma stimulates human macrophages to make 1,25-dihydroxy-vitaminD3. Similarly, IL-12 has been shown to play a role in stimulatingresistance to M. tuberculosis infection. For a review of the immunologyof M. tuberculosis infection, see Chan and Kaufmann, in Tuberculosis:Pathogenesis, Protection and Control, Bloom (ed.), ASM Press.Washington, D.C. (1994).

Existing compounds and methods for diagnosing tuberculosis or forinducing protective immunity against tuberculosis include the use ofpolypeptides that contain at least one immunogenic portion of one ormore Mycobacterium proteins and DNA molecules encoding suchpolypeptides. Diagnostic kits containing such polypeptides or DNAsequences and a suitable detection reagent may be used for the detectionof Mycobacterium infection in patients and biological samples.Antibodies directed against such polypeptides are also provided. Inaddition, such compounds may be formulated into vaccines and/orpharmaceutical compositions for immunization against Mycobacteriuminfection. (U.S. Pat. Nos. 6,949,246 and 6,555,653).

Malaria was eliminated in many parts of the world in the 1960s, but thedisease still persists and new strains of the disease are emerging thatare resistant to existing drugs. Malaria is a major public healthproblem in more than 90 countries. Nine out of ten cases of malariaoccur in sub-Saharan Africa. More than one third of the world'spopulation is at risk, and between 350 and 500 million people areinfected with malaria each year. Forty-five million pregnant women areat risk of contracting malaria this year. Of those individuals alreadyinfected, more than 1 million of those infected die each year from whatis a preventable disease. The majority of those deaths are children inAfrica.

Malaria is usually transmitted when a person is bitten by an infectedfemale Anopheles mosquito. To transmit the mosquito must have beeninfected by having drawn blood from a person already infected withmalaria. Malaria is caused by a parasite and the clinical symptoms ofthe disease include fever and flu-like illness, such as chills,headache, muscle aches, and tiredness. These symptoms may be accompaniedby nausea, vomiting, and diarrhea. Malaria can also cause anemia andjaundice because of the loss of red blood cells. Infection with one typeof malaria, Plasmodium falciparum, if not promptly treated, may causekidney failure, seizures, mental confusion, coma, and death.

An in vitro diagnostic method for malaria in an individual is known,comprising placing a tissue or a biological fluid taken from anindividual in contact with a molecule or polypeptide composition,wherein said molecule or polypeptide composition comprises one or morepeptide sequences bearing all or part of one or more T epitopes of theproteins resulting from the infectious activity of P. falciparum, underconditions allowing an in vitro immunological reaction to occur betweensaid composition and the antibodies that may be present in the tissue orbiological fluid, and in vitro detection of the antigen-antibodycomplexes formed (see, e.g., U.S. Pat. No. 7,087,231).

Expression and purification of a recombinant Plasmodium falciparum (3D7)AMA-1 ectodomain have been described. Previous methods have produced ahighly purified protein which retains folding and disulfide bridging ofthe native molecule. The recombinant AMA-1 is useful as a diagnosticreagentas well as in antibody production, and as a protein for usealone, or as part of, a vaccine to prevent malaria. (U.S. Pat. No.7,029,685)

Polynucleotides have been described in the art that encodespecies-specific P. vivax malarial peptide antigens which are proteinsor fragments of proteins secreted into the plasma of a susceptiblemammalian host after infection, as have monoclonal or polyclonalantibodies directed against these antigens. The peptide antigens,monoclonal antibodies, and/or polyclonal antibodies are utilized inassays used to diagnose malaria, as well as to determine whetherPlasmodium vivax is the species responsible for the infection. (U.S.Pat. No. 6,706,872) Species-specific P. vivax malarial peptide antigenshave also been reported which are proteins or fragments of proteinssecreted into the plasma of a susceptible mammalian host afterinfection, as have monoclonal or polyclonal antibodies directed againstthese antigens. The peptide antigens, monoclonal antibodies, and/orpolyclonal antibodies are utilized in assays used to diagnose malaria,as well as to determine whether Plasmodium vivax is the speciesresponsible for the infection (see, e.g., U.S. Pat. No. 6,231,861).

A recombinant Plasmodium falciparum (3D7) AMA-1 ectodomain has also beenexpressed by a method that produces a highly purified protein whichretains folding and disulfide bridging of the native molecule. Therecombinant AMA-1 is useful as a diagnostic reagent, for use in antibodyproduction, and as a vaccine. (U.S. Pat. No. 7,060,276) Similarly knownare the expression and purification of a recombinant Plasmodiumfalciparum (3D7) MSP-1₄₂, which retains folding and disulfide bridgingof the native molecule. The recombinant MSP-1₄₂ is useful as adiagnostic reagent, for use in antibody production, and as a vaccine.(U.S. Pat. No. 6,855,322)

Diagnostic methods for the detection of human malaria infections toidentify a subject having or suspected of being at risk for having aninfection with a malaria infectious pathogen are thus known according tothese and related disclosures. Specifically, for example, blood samplesare combined with a reagent containing 3-acetyl pyridine adeninedinucleotide (APAD), a substrate (e.g. a lactate salt or lactic acid),and a buffer. The reagent is designed to detect the presence of a uniqueglycolytic enzyme produced by the malaria parasite. This enzyme is knownas parasite lactic acid dehydrogenase (PLDH). PLDH is readilydistinguishable from host LDH using the above-described reagent.Combination of the reagent with a parasitized blood sample results inthe reduction of APAD. However, APAD is not reduced by host LDH. Thereduced APAD may then be detected by various techniques, includingspectral, fluorimetric, electrophoretic, or colorimetric analysis.Detection of the reduced APAD in the foregoing manner provides apositive indication of malaria infection (e.g., U.S. Pat. No.5,124,141). In another methodology for diagnosing malaria, a polypeptidecomprising a characteristic amino acid sequence derived from thePlasmodium falciparum antigen GLURP, is recognized in a test sample by aspecific antibody raised against or reactive with the polypeptide. (U.S.Pat. No. 5,231,168)

Leishmaniasis is a widespread parasitic disease with frequent epidemicsin the Indian subcontinent, Africa, and Latin America and is a WorldHealth Organization priority for vaccine development. A complex ofdifferent diseases, Leishmania parasites cause fatal infections ofinternal organs, as well as serious skin disease. One of the mostdevastating forms of leishmaniasis is a disfiguring infection of thenose and mouth. The number of cases of leishmaniasis is increasing, andit is now out of control in many areas. Leishmaniasis is also on therise in some developed countries, specifically southern Europe as aresult of HIV infection. Available drugs are toxic, expensive, andrequire long-term daily injections.

Leishmania are protozoan parasites that inhabit macrophages or the whiteblood cells of the immune system. The parasites are transmitted by thebite of small blood sucking insects (sand flies), which are difficult tocontrol, as they inhabit vast areas of the planet.

Visceral leishmaniasis is the most dangerous of the three manifestationsof the disease. It is estimated that about 500,000 new cases of thevisceral form (kala-azar or “the killing disease”) occur each year. Morethan 200 million people are currently at risk for contracting visceralleishmaniasis. Over 90 percent of visceral leishmaniasis cases occur inIndia, Bangladesh, Sudan, Brazil, and Nepal. Most of the deaths occur inchildren. Those with the cutaneous forms are often left permanentlydisfigured.

Leishmania infections are difficult to diagnose and typically involvehistopathologic analysis of tissue biopsy specimens. Several serologicaland immunological diagnostic assays have, however, been developed. (U.S.Pat. No. 7,008,774; Senaldi et al., (1996) J. Immunol. Methods 193:9 5;Zijlstra, et al., (1997) Trans. R. Soc. Trop. Med. Hyg. 91:671 673;Badaro, et al., (1996) J. Inf. Dis. 173:758 761; Choudhary, S., et al.,(1992) J. Comm. Dis. 24:32 36; Badaro, R., et al., (1986) Am. J. Trop.Med. Hyg. 35:72 78; Choudhary, A., et al., (1990) Trans. R. Soc. Trop.Med. Hyg. 84:363 366; and Reed, S. G., et al., (1990) Am. J. Trop. Med.Hyg. 43:632 639). The promastigotes release metabolic products into theculture medium to produce conditioned medium. These metabolic productsare immunogenic to the host. See Schnur, L. F., et al., (1972) Isrl. J.Med. Sci. 8:932 942; Sergeiev, V. P., et al., (1969) Med. Parasitol.38:208 212; El-On, J., et al., (1979) Exper. Parasitol. 47:254 269; andBray, R. S., et al., (1966) Trans. R. Soc. Trop. Med. Hyg. 60:605 609;U.S. Pat. No. 6,846,648, U.S. Pat. No. 5,912,166; U.S. Pat. No.5,719,263; U.S. Pat. No. 5,411,865).

About 40 million people around the world are infected with HIV, thevirus that causes AIDS. Around 3 million people die of the disease eachyear, 95 percent of them in the developing world. Each year, close to 5million people become infected with HIV. Currently, sub-Saharan Africancarries the highest burden of disease, but it is quickly spreading toother countries such as India, China, and Russia. The epidemic isgrowing most rapidly among minority populations. In the United Statesthere have been more than 950,000 cases of AIDS reported since 1981.AIDS hits people during their most productive years. Women, for bothbiological and social reasons, have an increased risk for HIV/AIDS.

AIDS is caused by human immunodeficiency virus (HIV), which kills anddamages cells of the body's immune system and progressively destroys thebody's ability to fight infections and certain cancers. HIV is spreadmost commonly by having unprotected sex with an infected partner. Themost robust solution to the problem is preventing the virus fromspreading. Making a safe, effective, and affordable HIV vaccine is oneway to reach this goal. Across the world, fewer than one in five peopleat high risk for HIV infection have access to effective prevention.

Methods for diagnosing HIV infections are known, including by virusculture, PCR of definitive nucleic acid sequences from patientspecimens, and antibody tests for the presence of anti-HIV antibodies inpatient sera, (see e.g., U.S. Pat. Nos. 6,979,535, 6,544,728, 6,316,183,6,261,762, 4,743,540.)

According to certain other embodiments as disclosed herein, the vaccinecompositions and related formulations and methods of use may include anantigen that is derived from a cancer cell, as may be useful for theimmunotherapeutic treatment of cancers. For example, the adjuvantformulation may finds utility with tumor rejection antigens such asthose for prostate, breast, colorectal, lung, pancreatic, renal ormelanoma cancers. Exemplary cancer or cancer cell-derived antigensinclude MACE 1, 3 and MACE 4 or other MACE antigens such as thosedisclosed in WO99/40188, PRAME, BAGE, Lage (also known as NY Eos 1) SAGEand HAGE (WO 99/53061) or GAGE (Robbins and Kawakami, 1996 CurrentOpinions in immunology 8, pps 628-636; Van den Eynde et al.,International Journal of Clinical & Laboratory Research (1997 & 1998);Correale et al. (1997), Journal of the National Cancer Institute 89, p.293. These non-limiting examples of cancer antigens are expressed in awide range of tumor types such as melanoma, lung carcinoma, sarcoma andbladder carcinoma. See, e.g., U.S. Pat. No. 6,544,518.

Other tumor-specific antigens are suitable for use with GLA according tocertain presently disclosed embodiments include, but are not restrictedto, tumor-specific or tumor-associated gangliosides such as GM₂, and GM₃or conjugates thereof to carrier proteins; or an antigen for use in aGLA vaccine composition for eliciting or enhancing an anti-cancer immuneresponse may be a self peptide hormone such as whole lengthGonadotrophin hormone releasing hormone (GnRH, WO 95/20600), a short 10amino acid long peptide, useful in the treatment of many cancers. Inanother embodiment prostate antigens are used, such as Prostate specificantigen (PSA), PAP, PSCA (e.g., Proc. Nat. Acad. Sci. USA 95(4)1735-1740 1998), PSMA or, in a preferred embodiment an antigen known asProstase. (e.g., Nelson, et al., Proc. Natl. Acad. Sci. USA (1999) 96:3114-3119; Ferguson, et al. Proc. Natl. Acad. Sci. USA 1999. 96,3114-3119; WO 98/12302; U.S. Pat. No. 5,955,306; WO 98/20117; U.S. Pat.Nos. 5,840,871 and 5,786,148; WO 00/04149. Other prostate specificantigens are known from WO 98/137418, and WO/004149. Another is STEAP(PNAS 96 14523 14528 7-12 1999).

Other tumor associated antigens useful in the context of the presentinvention include: Plu-1 (J Biol. Chem 274 (22) 15633-15645, 1999),HASH-1, HasH-2, Cripto (Salomon et al Bioessays 199, 21:61-70, U.S. Pat.No. 5,654,140) and Criptin (U.S. Pat. No. 5,981,215). Additionally,antigens particularly relevant for vaccines in the therapy of canceralso comprise tyrosinase and survivin.

The herein disclosed embodiments pertaining to GLA-containing vaccinecompositions comprising a cancer antigen will be useful against anycancer characterized by tumor associated antigen expression, such asHER-2/neu expression or other cancer-specific or cancer-associatedantigens.

Diagnosis of cancer in a subject having or suspected of being at riskfor having cancer may be accomplished by any of a wide range ofart-accepted methodologies, which may vary depending on a variety offactors including clinical presentation, degree of progression of thecancer, the type of cancer, and other factors. Examples of cancerdiagnostics include histopathological, histocytochemical,immunohistocytochemical and immunohistopathological examination ofpatient samples (e.g., blood, skin biopsy, other tissue biopsy, surgicalspecimens, etc.), PCR tests for defined genetic (e.g., nucleic acid)markers, serological tests for circulating cancer-associated antigens orcells bearing such antigens, or for antibodies of defined specificity,or other methodologies with which those skilled in the art will befamiliar. See, e.g., U.S. Pat. Nos. 6,734,172; 6,770,445; 6,893,820;6,979,730; 7,060,802; 7,030,232; 6,933,123; 6,682,901; 6,587,792;6,512,102; 7,078,180; 7,070,931; JP5-328975; Waslylyk et al., 1993 Eur.J Bioch. 211(7):18.

Vaccine compositions and methods according to certain embodiments of thepresent invention may also be used for the prophylaxis or therapy ofautoimmune diseases, which include diseases, conditions or disorderswherein a host's or subject's immune system detrimentally mediates animmune response that is directed against “self” tissues, cells,biomolecules (e.g., peptides, polypeptides, proteins, glycoproteins,lipoproteins, proteolipids, lipids, glycolipids, nucleic acids such asRNA and DNA, oligosaccharides, polysaccharides, proteoglycans,glycosaminoglycans, or the like, and other molecular components of thesubjects cells and tissues) or epitopes (e.g., specific immunologicallydefined recognition structures such as those recognized by an antibodyvariable region complementarity determining region (CDR) or by a T cellreceptor CDR.

Autoimmune diseases are thus characterized by an abnormal immuneresponse involving either cells or antibodies, that are in either casedirected against normal autologous tissues. Autoimmune diseases inmammals can generally be classified in one of two different categories:cell-mediated disease (i.e., T-cell) or antibody-mediated disorders.Non-limiting examples of cell-mediated autoimmune diseases includemultiple sclerosis, rheumatoid arthritis, Hashimoto thyroiditis, type Idiabetes mellitus (Juvenile onset diabetes) and autoimmune uvoretinitis.Antibody-mediated autoimmune disorders include, but are not limited to,myasthenia gravis, systemic lupus erythematosus (or SLE), Graves'disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia,autoimmune asthma, cryoglobulinemia, thrombic thrombocytopenic purpura,primary biliary sclerosis and pernicious anemia. The antigen(s)associated with: systemic lupus erythematosus is small nuclearribonucleic acid proteins (snRNP); Graves' disease is the thyrotropinreceptor, thyroglobulin and other components of thyroid epithelial cells(Akamizu et al., 1996; Kellerman et al., 1995; Raju et al., 1997; andTexier et al., 1992); pemphigus is cadherin-like pemphigus antigens suchas desmoglein 3 and other adhesion molecules (Memar et al., 1996:Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993); and thrombicthrombocytopenic purpura is antigens of platelets. (See, e.g., U.S. Pat.No. 6,929,796; Gorski et al. (Eds.), Autoimmunity, 2001, Kluwer AcademicPublishers, Norwell, Mass.; Radbruch and Lipsky, P. E. (Eds.) CurrentConcepts in Autoimmunity and Chronic Inflammation (Curr. Top. Microbial,and Immunol.) 2001, Springer, N.Y.)

Autoimmunity plays a role in more than 80 different diseases, includingtype 1 diabetes, multiple sclerosis, lupus, rheumatoid arthritis,scleroderma, and thyroid diseases. Vigorous quantitative estimates ofmorbidity for most autoimmune diseases are lacking. Most recent studiesdone in the late 1990s reveal that autoimmune diseases are the thirdmost common major illness in the United States; and the most commonautoimmune diseases affect more than 8.5 million Americans. Currentestimates of the prevalence of the disease range from 5 to 8 percent ofthe United States population. Most autoimmune diseasesdisproportionately affect women. Women are 2.7 times more likely thanmen to acquire an autoimmune disease. Women are more susceptible toautoimmune diseases; men appear to have higher levels of natural killercell activity than do women. (Jacobsen et al, Clinical Immunology andImmunopathology, 84:223-243, 1997.)

Autoimmune diseases occur when the immune system mistakes self tissuesfor nonself and mounts an inappropriate attack. The body can be affectedin different ways from autoimmune diseases, including, for example, thegut (Crohn's disease) and the brain (multiple sclerosis). It is knownthat an autoantibody attacks self-cells or self-tissues to injure theirfunction and as a result causes autoimmune diseases, and that theautoantibody may be detected in the patient's serum prior to the actualoccurrence of an autoimmune disease (e.g., appearance of clinical signsand symptoms). Detection of an autoantibody thus permits early discoveryor recognition of presence or risk for developing an autoimmune disease.Based on these findings, a variety of autoantibodies againstautoantigens have been discovered and the autoantibodies againstautoantigens have been measured in clinical tests (e.g., U.S. Pat. Nos.6,919,210, 6,596,501, 7,012,134, 6,919,078) while other autoimmunediagnostics may involve detection of a relevant metabolite (e.g., U.S.Pat. No. 4,659,659) or immunological reactivity (e.g., U.S. Pat. Nos.4,614,722 and 5,147,785, 4,420,558, 5,298,396, 5,162,990, 4,420,461,4,595,654, 5,846,758, 6,660,487).

In certain embodiments, the compositions of the invention will beparticularly applicable in treatment of the elderly and/or theimmunosuppressed, including subjects on kidney dialysis, subjects onchemo-therapy and/or radiation therapy, transplant recipients, and thelike. Such individuals generally exhibit diminished immune responses tovaccines and therefore use of the compositions of the invention canenhance the immune responses achieved in these subjects.

In other embodiments, the antigen or antigens used in the compositionsof the invention include antigens associated with respiratory diseases,such as those caused or exacerbated by bacterial infection (e.g.pneumococcal), for the prophylaxis and therapy of conditions such aschronic obstructive pulmonary disease (COPD). COPD is definedphysiologically by the presence of irreversible or partially reversibleairway obstruction in patients with chronic bronchitis and/or emphysema(Am J Respir Crit Care Med. 1995 November; 152(5 Pt 2):S77-121).Exacerbations of COPD are often caused by bacterial (e.g. pneumococcal)infection (Clin Microbiol Rev. 2001 April; 14(2):336-63). In aparticular embodiment, a composition of the invention comprises a GLAadjuvant, as described herein, in combination with the Pneumococcalvaccine Prevnar® (Wyeth).

In still other embodiments, the compositions of the invention,comprising GLA as described herein, are used in the treatment ofallergic conditions. For example, in a particular embodiment, thecompositions are used in allergy desensitization therapy. Such therapyinvolves the stimulation of the immune system with gradually increasingdoses of the substances to which a person is allergic, wherein thesubstances are formulated in compositions comprising GLA. In specificembodiments, the compositions are used in the treatment of allergies tofood products, pollen, mites, cats or stinging insects (e.g., bees,hornets, yellow jackets, wasps, velvet ants, fire ants).

TLR

As described herein, certain embodiments of the present inventioncontemplate vaccine compositions and immunological adjuvantcompositions, including pharmaceutical compositions, that include, inaddition to the GLA compound(s) of the invention, one or more toll-likereceptor agonist (TLR agonist). Toll-like receptors (TLR) include cellsurface transmembrane receptors of the innate immune system that conferearly-phase recognition capability to host cells for a variety ofconserved microbial molecular structures such as may be present in or ona large number of infectious pathogens. (e.g., Armant et al., 2002Genome Biol. 3(8): reviews 3011.1-3011.6; Fearon et al., 1996 Science272:50; Medzhitov et al., 1997 Curr. Opin. Immunol. 9:4; Luster 2002Curr. Opin. Immunol. 14:129; Lien et al. 2003 Nat. Immunol. 4:1162;Medzhitov, 2001 Nat. Rev. Immunol. 1:135; Takeda et al., 2003 Ann RevImmunol. 21:335: Takeda et al. 2005 Int. Immunol. 17:1; Kaisho et al.,2004 Microbes Infect. 6:1388; Datta et al., 2003 J. Immunol. 170:4102).

induction of TLR-mediated signal transduction to potentiate theinitiation of immune responses via the innate immune system may beeffected by TLR agonists, which engage cell surface TLR. For example,lipopolysaccharide (LPS) may be a TLR agonist through TLR2 or TLR4 (Tsanet al., 2004 J. Leuk. Biol. 76:514; Tsan et al., 2004 Am. J. Physiol.Cell Physiol. 286:C739; Lin et al., 2005 Shock 24:206);poly(inosine-cytidine) (polyl:C) may be a TLR agonist through TLR3(Salem et al., 2006 Vaccine 24:5119); CpG sequences(oligodeoxynucleotides containing unmethylated cytosine-guanosine or“CpG” dinucleotide motifs, e.g., CpG 7909, Cooper et al., 2005 ADS19:1473; CpG 10101 Bayes et al. Methods Find Exp Clin Pharmacol 27:193;Vollmer et al. Expert Opinion on Biological Therapy 5:673; Vollmer etal., 2004 Antimicrob. Agents Chemother. 48:2314; Deng et al., 2004 J.Immunol. 173:5148) may be TLR agonists through TLR9 (Andaloussi et a.,2006 Glia 54:526; Chen et al., 2006 J. Immunol. 177:2373);peptidoglycans may be TLR2 and/or TLR6 agonists (Soboll et al., 2006Biol. Reprod. 75:131; Nakao et al., 2005 J. Immunol. 174:1566); 3M003(4-amino-2-(ethoxymethyl)-α,α-dimethyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1-ethanolhydrate, Mol. Wt. 318 Da from 3M Pharmaceuticals, St. Paul, Minn., whichis also a source of the related compounds 3M001 and 3M002; Gorden etal., 2005 J. Immunol. 174:1259) may be a TLR7 agonist (Johansen 2005Clin. Exp. Allerg. 35:1591) and/or a TLR8 agonist (Johansen 2005);flagellin may be a TLR5 agonist (Feuillet et al., 2006 Proc. Nat. Acad.Sci. USA 103:12487); and hepatitis C antigens may act as TLR agoniststhrough TLR7 and/or TLR9 (Lee et al., 2006 Proc. Nat. Acad. Sci. USA103:1828; Horsmans et al., 2005 Hepatol. 42:724). Other TLR agonists areknown (e.g., Schirmbeck et al., 2003 J. Immunol. 171:5198) and may beused according to certain of the presently described embodiments.

For example, and by way of background (see, e.g., U.S. Pat. No.6,544,518) immunostimulatory oligonucleotides containing unmethylatedCpG dinucleotides (“CpG”) are known as being adjuvants when administeredby both systemic and mucosal routes (WO 96/02555, EP 468520, Davis etal., J. Immunol, 1998. 160(2):870-876; McCluskie and Davis, J. Immunol.,1998, 161(9):4463-6). CpG is an abbreviation for cytosine-guanosinedinucleotide motifs present in DNA. The central role of the CG motif inimmunostimulation was elucidated by Krieg, Nature 374, p 546 1995.Detailed analysis has shown that the CG motif has to be in a certainsequence context, and that such sequences are common in bacterial DNAbut are rare in vertebrate DNA. The immunostimulatory sequence is often:Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the dinucleotideCG motif is not methylated, but other unmethylated CpG sequences areknown to be immunostimulatory and may be used in certain embodiments ofthe present invention. CpG when formulated into vaccines, may beadministered in free solution together with free antigen (WO 96/02555;McCluskie and Davis, supra) or covalently conjugated to an antigen (POTPublication No. WO 98/16247), or formulated with a carrier such asaluminium hydroxide (e.g., Davis et al. supra, Brazolot-Millan et al.,Proc. Natl. Acad. Sci., USA, 1998, 95(26), 15553-8).

The preferred oligonucleotides for use in adjuvants or vaccines of thepresent invention preferably contain two or more dinucleotide CpG motifsseparated by at least three, more preferably at least six or morenucleotides. The oligonucleotides of the present invention are typicallydeoxynucleotides. In a preferred embodiment the internucleotide in theoligonucleotide is phosphorodithioate, or more preferably aphosphorothioate bond, although phosphodiester and other internucleotidebonds are within the scope of the invention including oligonucleotideswith mixed internucleotide linkages. Methods for producingphosphorothioate oligonucleotides or phosphorodithioate are described inU.S. Pat. Nos. 5,666,153, 5,278,302 and WO95/26204.

Examples of preferred oligonucleotides have sequences that are disclosedin the following publications; for certain herein disclosed embodimentsthe sequences preferably contain phosphorothioate modifiedinternucleotide linkages:

CPG 7909: Cooper et al., “CPG 7909 adjuvant improves hepatitis B virusvaccine seroprotection in antiretroviral-treated HIV-infected adults.”AIDS, 2005 Sep. 23; 19(14):1473-9.

CpG 10101: Bayes et al., “Gateways to clinical trials.” Methods Find.Exp. Clin. Pharmacol. 2005 April; 27(3):193-219.

Vollmer J., “Progress in drug development of immunostimulatory CpGoligodeoxynucleotide ligands for TLR9.” Expert Opinion on BiologicalTherapy. 2005 May; 5(5): 673-682

Alternative CpG oligonucleotides may comprise variants of the preferredsequences described in the above-cited publications that differ in thatthey have inconsequential nucleotide sequence substitutions, insertions,deletions and/or additions thereto. The CpG oligonucleotides utilized incertain embodiments of the present invention may be synthesized by anymethod known in the art (e.g., EP 468520). Conveniently, sucholigonucleotides may be synthesized utilizing an automated synthesizer.The oligonucleotides are typically deoxynucleotides. In a preferredembodiment the internucleotide bond in the oligonucleotide isphosphorodithioate, or more preferably phosphorothioate bond, althoughphosphodiesters are also within the scope of the presently contemplatedembodiments. Oligonucleotides comprising different internucleotidelinkages are also contemplated, e.g., mixed phosphorothioatephosphodiesters. Other internucleotide bonds which stabilize theoligonucleotide may also be used.

Co-Adjuvant

Certain embodiments as provided herein include vaccine compositions andimmunological adjuvant compositions, including pharmaceuticalcompositions, that contain, in addition to GLA compound(s), at least oneco-adjuvant, which refers to a component of such compositions that hasadjuvant activity but that is other than GLA. A co-adjuvant having suchadjuvant activity includes a composition that, when administered to asubject such as a human (e.g., a human patient), a non-human primate, amammal or another higher eukaryotic organism having a recognized immunesystem, is capable of altering (i.e., increasing or decreasing in astatistically significant manner, and in certain preferred embodiments,enhancing or increasing) the potency and/or longevity of an immuneresponse. (See, e.g., Powell and Newman, “Vaccine design—The Subunit andAdjuvant Approach”, 1995, Plenum Press, New York) In certain embodimentsdisclosed herein GLA and a desired antigen, and optionally one or moreco-adjuvants, may so alter, e.g., elicit or enhance, an immune responsethat is directed against the desired antigen which may be administeredat the same time as GLA or may be separated in time and/or space (e.g.,at a different anatomic site) in its administration, but certaininvention embodiments are not intended to be so limited and thus alsocontemplate administration of GLA in a composition that does not includea specified antigen but which may also include one or more of a TLRagonist, a co-adjuvant, an imidazoquinline immune response modifier, anda double stem loop immune modifier (dSLIM).

Accordingly and as noted above, co-adjuvants include compositions otherthan GLA that have adjuvant effects, such as saponins and saponinmimetics, including QS21 and QS21 mimetics (see, e.g., U.S. Pat. No.5,057,540; EP 0 362 279 B1; WO 95/17210), alum, plant alkaloids such astomatine, detergents such as (but not limited to) saponin, polysorbate80, Span 85 and stearyl tyrosine, one or more cytokines (e.g., GM-CSF,IL-2, IL-7, IL-12, TNF-alpha, IFN-gamma), an imidazoquinoline immuneresponse modifier, and a double stem loop immune modifier (dSLIM, e.g.,Weeratna et al., 2005 Vaccine 23:5263).

Detergents including saponins are taught in, e.g., U.S. Pat. No.6,544,518; Lacaille-Dubois, M and Wagner H. (1996 Phytomedicine2:363-386), U.S. Pat. No. 5,057,540, Kensil, Crit Rev Ther Drug CarrierSyst, 1996, 12 (1-2):1-55, and EP 0 362 279 B1. Particulate structures,termed Immune Stimulating Complexes (ISCOMS), comprising fractions ofQuil A (saponin) are haemolytic and have been used in the manufacture ofvaccines (Morein, B., EP 0 109 942 B1). These structures have beenreported to have adjuvant activity (EP 0 109 942 B1; WO 96/11711). Thehaemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A)have been described as potent systemic adjuvants, and the method oftheir production is disclosed in U.S. Pat. No. 5,057,540 and EP 0 362279 B1. Also described in these references is the use of QS7 (anon-haemolytic fraction of Quil-A) which acts as a potent adjuvant forsystemic vaccines. Use of QS21 is further described in Kensil et al.(1991. J. Immunology 146:431-437). Combinations of QS21 and polysorbateor cyclodextrin are also known (WO 99/10008). Particulate adjuvantsystems comprising fractions of QuilA, such as QS21 and QS7 aredescribed in WO 96/33739 and WO 96/11711. Other saponins which have beenused in systemic vaccination studies include those derived from otherplant species such as Gypsophila and Saponaria (Bomford et al., Vaccine,10(9):572-577, 1992).

Escin is another detergent related to the saponins for use in theadjuvant compositions of the embodiments herein disclosed. Escin isdescribed in the Merck index (12^(th) Ed.: entry 3737) as a mixture ofsaponin occurring in the seed of the horse chestnut tree, Aesculushippocastanum. Its isolation is described by chromatography andpurification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and byion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractionsof escin (also known as aescin) have been purified and shown to bebiologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996August; 44(8): 1454-1464)). Digitonin is another detergent, also beingdescribed in the Merck index (12th Ed., entry 3204) as a saponin, beingderived from the seeds of Digitalis purpurea and purified according tothe procedure described by Gisvold et al., J. Am. Pharm. Assoc., 1934,23, 664; and Rubenstroth-Bauer, Physiol. Chem., 1955, 301, 621.

Other co-adjuvants for use according to certain herein disclosedembodiments include a block co-polymer or biodegradable polymer, whichrefers to a class of polymeric compounds with which those in therelevant art will be familiar. Examples of a block co-polymer orbiodegradable polymer that may be included in a GLA vaccine compositionor a GLA immunological adjuvant include Pluronic® L121 (BASF Corp.,Mount Olive, N.J.; see, e.g., Yeh et al., 1996 Pharm. Res. 13:1693; U.S.Pat. No. 5,565,209), CRL1005 (e.g., Triozzi et al., 1997 Clin Canc. Res.3:2355), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA),poly-(D,L-lactide-co-glycolide) (PLG), and polyl:C. (See, e.g., Powelland Newman, “Vaccine design—The Subunit and Adjuvant Approach”, 1995,Plenum Press, New York)

Certain embodiments contemplate GLA vaccines and GLA immunologicaladjuvants that include an oil, which in some such embodiments maycontribute co-adjuvant activity and in other such embodiments mayadditionally or alternatively provide a pharmaceutically acceptablecarrier or excipient. Any number of suitable oils are known and may beselected for inclusion in vaccine compositions and immunologicaladjuvant compositions based on the present disclosure. Examples of suchoils, by way of illustration and not limitation, include squalene,squalane, mineral oil, olive oil, cholesterol, and a mannide monooleate.

Immune response modifiers such as imidazoquinoline immune responsemodifiers are also known in the art and may also be included asco-adjuvants in certain presently disclosed embodiments. Certainpreferred imidazoquinoline immune response modifiers include, by way ofnon-limiting example, resiquimod (R848), imiquimod and gardiquimod(Hemmi et al., 2002 Nat. Immunol. 3:196; Gibson et al., 2002 Cell.Immunol. 218:74; Gorden et al., 2005 J. Immunol. 174:1259); these andother imidazoquinoline immune response modifiers may, under appropriateconditions, also have TLR agonist activity as described herein. Otherimmune response modifiers are the nucleic acid-based double stem loopimmune modifiers (dSLIM). Specific examples of dSLIM that arecontemplated for use in certain of the presently disclosed embodimentscan be found in Schmidt et al., 2006 Allergy 61:56; Weihrauch et al.2005 Clin Cancer Res. 11(16):5993-6001; Modern Biopharmaceuticals, J.Knäblein (Editor). John Wiley & Sons, Dec. 6, 2005. (dSLIM discussed onpages 183 to ˜200), and from Mologen AG (Berlin, FRG: [retrieved onlineon Aug. 18, 2006 at http://www.mologen.com/English/04.20-dSLIM.shtml].

As also noted above, one type of co-adjuvant for use with GLA asdescribed herein may be the aluminum co-adjuvants, which are generallyreferred to as “alum.” Alum co-adjuvants are based on the following:aluminum oxy-hydroxide; aluminum hydroxyphosphoate; or variousproprietary salts. Vaccines that use alum co-adjuvants may includevaccines for tetanus strains, HPV, hepatitis A, inactivated polio virus,and other antigens as described herein. Alum co-adjuvants areadvantageous because they have a good safety record, augment antibodyresponses, stabilize antigens, and are relatively simple for large-scaleproduction. (Edelman 2002 Mol. Biotechnol. 21:129-148; Edelman, R. 1980Rev. Infect. Dis. 2:370-383.)

Other co-adjuvants that may be combined with GLA for effective immunestimulation include saponins and saponin mimetics, including QS21 andstructurally related compounds conferring similar effects and referredto herein as QS21 mimetics. QS21 has been recognized as a preferredco-adjuvant. QS21 may comprise an HPLC purified non-toxic fractionderived from the bark of Quillaja Saponaria Molina. The production ofQS21 is disclosed in U.S. Pat. No. 5,057,540. (See also U.S. Pat. Nos.6,936,255, 7,029,678 and 6,932,972.)

GLA may also in certain embodiments be combined with “immunostimulatorycomplexes” known as ISCOMS (e.g., U.S. Pat. Nos. 6,869,607, 6,846,489,6,027,732, 4,981,684), including saponin-derived ISCOMATRIX®, which iscommercially available, for example, from Iscotec (Stockholm, Sweden)and CSL Ltd. (Parkville, Victoria, Australia).

Recombinant Expression Construct

According to certain herein disclosed embodiments, the GLA vaccinecomposition may contain at least one recombinant expression constructwhich comprises a promoter operably linked to a nucleic acid sequenceencoding an antigen. In certain further embodiments the recombinantexpression construct is present in a viral vector, such as anadenovirus, adeno-associated virus, herpesvirus, lentivirus, poxvirus orretrovirus vector. Compositions and methods for making and using suchexpression constructs and vectors are known in the art, for theexpression of polypeptide antigens as provided herein, for example,according to Ausubel et al. (Eds.), Current Protocols in MolecularBiology, 2006 John Wiley & Sons, NY. Non-limiting examples ofrecombinant expression constructs generally can be found, for instance,in U.S. Pat. Nos. 6,844,192; 7,037,712; 7,052,904; 7,001,770; 6,106,824;5,693,531; 6,613,892; 6,875,610; 7,067,310; 6,218,186; 6,783,981;7,052,904; 6,783,981; 6,734,172; 6,713,068; 5,795,577 and 6,770,445 andelsewhere, with teachings that can be adapted to the expression ofpolypeptide antigens as provided herein, for use in certain presentlydisclosed embodiments.

Immune Response

The invention thus provides compositions for altering (i.e., increasingor decreasing in a statistically significant manner, for example,relative to an appropriate control as will be familiar to personsskilled in the art) immune responses in a host capable of mounting animmune response. As will be known to persons having ordinary skill inthe art, an immune response may be any active alteration of the immunestatus of a host, which may include any alteration in the structure orfunction of one or more tissues, organs, cells or molecules thatparticipate in maintenance and/or regulation of host immune status.Typically, immune responses may be detected by any of a variety of wellknown parameters, including but not limited to in vivo or in vitrodetermination of: soluble immunoglobulins or antibodies; solublemediators such as cytokines, lymphokines, chemokines, hormones, growthfactors and the like as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators; cellular activationstate changes as determined by altered functional or structuralproperties of cells of the immune system, for example cellproliferation, altered motility, induction of specialized activitiessuch as specific gene expression or cytolytic behavior; cellulardifferentiation by cells of the immune system, including altered surfaceantigen expression profiles or the onset of apoptosis (programmed celldeath); or any other criterion by which the presence of an immuneresponse may be detected.

Immune responses may often be regarded, for instance, as discriminationbetween self and non-self structures by the cells and tissues of ahost's immune system at the molecular and cellular levels, but theinvention should not be so limited. For example, immune responses mayalso include immune system state changes that result from immunerecognition of self molecules, cells or tissues, as may accompany anynumber of normal conditions such as typical regulation of immune systemcomponents, or as may be present in pathological conditions such as theinappropriate autoimmune responses observed in autoimmune anddegenerative diseases. As another example, in addition to induction byup-regulation of particular immune system activities (such as antibodyand/or cytokine production, or activation of cell mediated immunity)immune responses may also include suppression, attenuation or any otherdown-regulation of detectable immunity, which may be the consequence ofthe antigen selected, the route of antigen administration, specifictolerance induction or other factors.

Determination of the induction of an immune response by the vaccines ofthe present invention may be established by any of a number of wellknown immunological assays with which those having ordinary skill in theart will be readily familiar. Such assays include, but need not belimited to, to in vivo or in vitro determination of: soluble antibodies;soluble mediators such as cytokines, lymphokines, chemokines, hormones,growth factors and the like as well as other soluble small peptide,carbohydrate, nucleotide and/or lipid mediators; cellular activationstate changes as determined by altered functional or structuralproperties of cells of the immune system, for example cellproliferation, altered motility, induction of specialized activitiessuch as specific gene expression or cytolytic behavior; cellulardifferentiation by cells of the immune system, including altered surfaceantigen expression profiles or the onset of apoptosis (programmed celldeath). Procedures for performing these and similar assays are widelyknown and may be found, for example in Lefkovits (Immunology MethodsManual: The Comprehensive Sourcebook of Techniques, 1998; see alsoCurrent Protocols in Immunology; see also, e.g., Weir, Handbook ofExperimental Immunology, 1986 Blackwell Scientific, Boston, Mass.;Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, 1979Freeman Publishing, San Francisco, Calif.; Green and Reed, 1998 Science281:1309 and references cited therein.).

Detection of the proliferation of antigen-reactive T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring the rate of DNA synthesis,and antigen specificity can be determined by controlling the stimuli(such as, for example, a specific desired antigen- or a controlantigen-pulsed antigen presenting cells) to which candidateantigen-reactive T cells are exposed. T cells which have been stimulatedto proliferate exhibit an increased rate of DNA synthesis. A typical wayto measure the rate of DNA synthesis is, for example, by pulse-labelingcultures of T cells with tritiated thymidine, a nucleoside precursorwhich is incorporated into newly synthesized DNA. The amount oftritiated thymidine incorporated can be determined using a liquidscintillation spectrophotometer. Other ways to detect T cellproliferation include measuring increases in interleukin-2 (IL-2)production, Ca²⁺ flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to a particular antigenmay be quantified.

Detection of antigen-specific antibody production may be achieved, forexample, by assaying a sample (e.g., an immunoglobulin containing samplesuch as serum, plasma or blood) from a host treated with a vaccineaccording to the present invention using in vitro methodologies such asradioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISA),equilibrium dialysis or solid phase immunoblotting including Westernblotting. In preferred embodiments ELISA assays may further includeantigen-capture immobilization of the target antigen with a solid phasemonoclonal antibody specific for the antigen, for example, to enhancethe sensitivity of the assay. Elaboration of soluble mediators (e.g.,cytokines, chemokines, lymphokines, prostaglandins, etc.) may also bereadily determined by enzyme-linked immunosorbent assay (ELISA), forexample, using methods, apparatus and reagents that are readilyavailable from commercial sources (e.g., Sigma, St. Louis, Mo.; see alsoR & D Systems 2006 Catalog, R & Systems, Minneapolis, Minn.).

Any number of other immunological parameters may be monitored usingroutine assays that are well known in the art. These may include, forexample, antibody dependent cell-mediated cytotoxicity (ADCC) assays,secondary in vitro antibody responses, flow immunocytofluorimetricanalysis of various peripheral blood or lymphoid mononuclear cellsubpopulations using well established marker antigen systems,immunohistochemistry or other relevant assays. These and other assaysmay be found, for example, in Rose et al. (Eds.), Manual of ClinicalLaboratory Immunology, 5^(th) Ed., 1997 American Society ofMicrobiology, Washington, D.C.

Accordingly it is contemplated that the vaccine and adjuvantcompositions provided herein will be capable of eliciting or enhancingin a host at least one immune response that is selected from aT_(H)1-type T lymphocyte response, a T_(H)2-type T lymphocyte response,a cytotoxic T lymphocyte (CTL) response, an antibody response, acytokine response, a lymphokine response, a chemokine response, and aninflammatory response. In certain embodiments the immune response maycomprise at least one of production of one or a plurality of cytokineswherein the cytokine is selected from interferon-gamma (IFN-γ), tumornecrosis factor-alpha (TNF-α), production of one or a plurality ofinterleukins wherein the interleukin is selected from IL-1, IL-2, IL-3,IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18 and IL-23,production one or a plurality of chemokines wherein the chemokine isselected from MIP-1α, MIP-1β, RANTES, CCL4 and CCL5, and a lymphocyteresponse that is selected from a memory T cell response, a memory B cellresponse, an effector T cell response, a cytotoxic T cell response andan effector B cell response. See, e.g., WO 94/00153; WO 95/17209; WO96/02555; U.S. Pat. No. 6,692,752; U.S. Pat. No. 7,084,256; U.S. Pat.No. 6,977,073; U.S. Pat. No. 6,749,856; U.S. Pat. No. 6,733,763; U.S.Pat. No. 6,797,276; U.S. Pat. No. 6,752,995; U.S. Pat. No. 6,057,427;U.S. Pat. No. 6,472,515; U.S. Pat. No. 6,309,847; U.S. Pat. No.6,969,704; U.S. Pat. No. 6,120,769; U.S. Pat. No. 5,993,800; U.S. Pat.No. 5,595,888; Smith et al., 1987 J Biol Chem. 262:6951; Kriegler etal., 1988 Cell 53:45 53; Beutler et al., 1986 Nature 320:584; U.S. Pat.No. 6,991,791; U.S. Pat. No. 6,654,462; U.S. Pat. No. 6,375,944.

Pharmaceutical Compositions

Pharmaceutical compositions generally comprise at least one GLA compoundof the invention, and may further comprise one or more components asprovided herein that are selected, for example, from antigen, TLRagonist, co-adjuvant (including optionally a cytokine, animidazoquinoline immune response modifier and/or a dSLIM), and/or arecombinant expression construct, in combination with a pharmaceuticallyacceptable carrier, excipient or diluent.

Therefore, in certain aspects, the present invention is drawn to GLA“monotherapy” wherein GLA, as described herein, is formulated in acomposition that is substantially devoid of other antigens, and isadministered to a subject in order to stimulate an immune e response,e.g., a non-specific immune response, for the purpose of treating orpreventing a disease or other condition, such as for treating aninfection by an organism, for treating seasonal rhinitis, or the like.In one embodiment, for example, the compositions and methods of theinvention employ a GLA compound for stimulating an immune response in asubject. In another embodiment, the GLA is in the form of a spray,optionally provided in a kit.

The GLA may be preferably formulated in a stable emulsion. In oneparticular embodiment, for example, a composition is provided comprisinga GLA compound of the invention in a stable emulsion substantiallydevoid of other antigens.

In certain other embodiments, the pharmaceutical composition is avaccine composition that comprises both GLA and an antigen and mayfurther comprise one or more components, as provided herein, that areselected from TLR agonist, co-adjuvant (including, e.g., a cytokine, animidazoquinoline immune response modifier and/or a dSLIM) and the likeand/or a recombinant expression construct, in combination with apharmaceutically acceptable carrier, excipient or diluent.

Illustrative carriers will be nontoxic to recipients at the dosages andconcentrations employed. For GLA-plus-nucleic acid-based vaccines, orfor vaccines comprising GLA plus an antigen, about 0.001 μg/kg to about100 mg/kg body weight will generally be administered, typically by theintradermal, subcutaneous, intramuscular or intravenous route, or byother routes.

In a more specific embodiment, the dosage is about 0.001 μg/kg to about1 mg/kg. In another specific embodiment, the dosage is about 0.001 toabout 50 μg/kg. In another specific embodiment, the dosage is about0.001 to about 15 μg/kg.

In another specific embodiment, the amount of GLA administered is about0.01 μg/dose to about 5 mg/dose. In another specific embodiment, theamount of GLA administered is about 0.1 μg/dose to about 1 mg/dose. Inanother specific embodiment, the amount of GLA administered is about 0.1μg/dose to about 100 μg/dose. In another specific embodiment, the GLAadministered is about 0.1 μg/dose to about 10 μg/dose.

It will be evident to those skilled in the art that the number andfrequency of administration will be dependent upon the response of thehost. “Pharmaceutically acceptable carriers” for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

“Pharmaceutically acceptable salt” refers to salts of the compounds ofthe present invention derived from the combination of such compounds andan organic or inorganic acid (acid addition salts) or an organic orinorganic base (base addition salts). The compositions of the presentinvention may be used in either the free base or salt forms, with bothforms being considered as being within the scope of the presentinvention.

The pharmaceutical compositions may be in any form which allows for thecomposition to be administered to a patient. For example, thecomposition may be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral (e.g., sublingually or buccally), sublingual,rectal, vaginal, and intranasal (e.g., as a spray). The term parenteralas used herein includes iontophoretic (e.g., U.S. Pat. Nos. 7,033,598;7,018,345; 6,970,739), sonophoretic (e.g., U.S. Pat. Nos. 4,780,212;4,767,402; 4,948,587; 5,618,275; 5,656,016; 5,722,397; 6,322,532;6,018,678), thermal (e.g., U.S. Pat. Nos. 5,885,211; 6,685,699), passivetransdermal (e.g., U.S. Pat. Nos. 3,598,122; 3,598,123; 4,286,592;4,314,557; 4,379,454; 4,568,343; 5,464,387; UK Pat. Spec. No. 2232892;U.S. Pat. Nos. 6,871,477; 6,974,588; 6,676,961), microneedle (e.g., U.S.Pat. Nos. 6,908,453; 5,457,041; 5,591,139; 6,033,928) administration andalso subcutaneous injections, intravenous, intramuscular, intrasternal,intracavernous, intrathecal, intrameatal, intraurethral injection orinfusion techniques. In a particular embodiment, a composition asdescribed herein (including vaccine and pharmaceutical compositions) isadministered intradermally by a technique selected from iontophoresis,microcavitation, sonophoresis or microneedles.

The pharmaceutical composition is formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa patient take the form of one or more dosage units, where for example,a tablet may be a single dosage unit, and a container of one or morecompounds of the invention in aerosol form may hold a plurality ofdosage units.

For oral administration, an excipient and/or binder may be present.Examples are sucrose, kaolin, glycerin, starch dextrins, sodiumalginate, carboxymethylcellulose and ethyl cellulose. Coloring and/orflavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred compositions contain one ormore of a sweetening agent, preservatives, dye/colorant and flavorenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

A liquid pharmaceutical composition as used herein, whether in the formof a solution, suspension or other like form, may include one or more ofthe following carriers or excipients: sterile diluents such as water forinjection, saline solution, preferably physiological saline, Ringer'ssolution, isotonic sodium chloride, fixed oils such as squalene,squalene, mineral oil, a mannide monooleate, cholesterol, and/orsynthetic mono or digylcerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. An injectable pharmaceutical composition ispreferably sterile.

In a particular embodiment, a pharmaceutical or vaccine composition ofthe invention comprises a stable aqueous suspension of less than 0.2 umand further comprises at least one component selected from the groupconsisting of phospholipids, fatty acids, surfactants, detergents,saponins, fluorodated lipids, and the like.

In another embodiment, a composition of the invention is formulated in amanner which can be aerosolized.

It may also be desirable to include other components in a vaccine orpharmaceutical composition, such as delivery vehicles including but notlimited to aluminum salts, water-in-oil emulsions, biodegradable oilvehicles, oil-in-water emulsions, biodegradable microcapsules, andliposomes. Examples of additional immunostimulatory substances(co-adjuvants) for use in such vehicles are also described above and mayinclude N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12,GM-CSF, gamma interferon and IL-12.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release is desired. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactic galactide) mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109. In this regard, itis preferable that the microsphere be larger than approximately 25microns.

Pharmaceutical compositions (including GLA vaccines and GLAimmunological adjuvants) may also contain diluents such as buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents. Preferably, product may be formulated as alyophilizate using appropriate excipient solutions (e.g., sucrose) asdiluents.

As described above, in certain embodiments the subject inventionincludes compositions capable of delivering nucleic acid moleculesencoding desired antigens. Such compositions include recombinant viralvectors (e.g., retroviruses (see WO 90/07936, WO 91/02805, WO 93/25234,WO 93/25698, and WO 94/03622), adenovirus (see Berkner, Biotechniques6:616-627, 1988; Li et al., Hum. Gene Ther. 4:403-409, 1993; Vincent etal., Nat. Genet. 5:130-134, 1993; and Kolls et al., Proc. Natl. Acad.Sci. USA 91:215-219, 1994), pox virus (see U.S. Pat. No. 4,769,330; U.S.Pat. No. 5,017,487; and WO 89/01973)), recombinant expression constructnucleic acid molecules complexed to a polycationic molecule (see WO93/03709), and nucleic acids associated with liposomes (see Wang et al.,Proc. Natl. Acad. Sci. USA 84:7851, 1987). In certain embodiments, theDNA may be linked to killed or inactivated adenovirus (see Curiel etal., Hum. Gene Ther. 3:147-154, 1992; Cotton et al., Proc. Natl. Acad.Sci. USA 89:6094, 1992). Other suitable compositions include DNA-ligand(see Wu et al., J. Biol. Chem. 264:16985-16987, 1989) and lipid-DNAcombinations (see Feigner et al., Proc. Natl. Acad. Sci. USA84:7413-7417, 1989).

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from a host, modified, and placed into thesame or another host animal. It will be evident that one can utilize anyof the compositions noted above for introduction of antigen-encodingnucleic acid molecules into tissue cells in an ex vivo context.Protocols for viral, physical and chemical methods of uptake are wellknown in the art.

Accordingly, the present invention is useful for enhancing or eliciting,in a host, a patient or in cell culture, an immune response. As usedherein, the term “patient” refers to any warm-blooded animal, preferablya human. A patient may be afflicted with an infectious disease, cancer,such as breast cancer, or an autoimmune disease, or may be normal (i.e.,free of detectable disease and/or infection). A “cell culture” is anypreparation containing immunocompetent cells or isolated cells of theimmune system (including, but not limited to, T cells, macrophages,monocytes, B cells and dendritic cells). Such cells may be isolated byany of a variety of techniques well known to those of ordinary skill inthe art (e.g., Ficoll-hypaque density centrifugation). The cells may(but need not) have been isolated from a patient afflicted with cancer,and may be reintroduced into a patient after treatment.

In certain embodiments a liquid composition intended for eitherparenteral or oral administration should contain an amount of GLAvaccine composition such that a suitable dosage will be obtained.Typically, this amount is at least 0.01 wt % of an antigen in thecomposition. When intended for oral administration, this amount may bevaried to be between 0.1 and about 70% of the weight of the composition.Preferred oral compositions contain between about 4% and about 50% ofthe antigen. Preferred compositions and preparations are prepared sothat a parenteral dosage unit contains between 0.01 to 1% by weight ofactive composition.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the antigen (e.g., GLA-antigen vaccine composition) orGLA (e.g., immunological adjuvant composition; GLA is available fromAvanti Polar Lipids, Inc., Alabaster, Ala.; e.g., product number 699800)of from about 0.1 to about 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol. In the methods of the invention, the vaccinecompositions/adjuvants may be administered through use of insert(s),bead(s), timed-release formulation(s), patch(es) or fast-releaseformulation(s).

Also contemplated in certain embodiments are kits comprising the hereindescribed GLA vaccine compositions and/or GLA immunological adjuvantcompositions, which may be provided in one or more containers. In oneembodiment all components of the GLA vaccine compositions and/or GLAimmunological adjuvant compositions are present together in a singlecontainer, but the invention embodiments are not intended to be solimited and also contemplate two or more containers in which, forexample, a GLA immunological adjuvant composition is separate from, andnot in contact with, the antigen component. By way of non-limitingtheory, it is believed that in some cases administration only of the GLAimmunological adjuvant composition may be performed beneficially, whilstin other cases such administration may beneficially be separatedtemporally and/or spatially (e.g., at a different anatomical site) fromadministration of the antigen, whilst in still other casesadministration to the subject is beneficially conducted of a GLA vaccinecomposition as described herein and containing both antigen and GLA, andoptionally other herein described components as well.

A container according to such kit embodiments may be any suitablecontainer, vessel, vial, ampule, tube, cup, box, bottle, flask, jar,dish, well of a single-well or multi-well apparatus, reservoir, tank, orthe like, or other device in which the herein disclosed compositions maybe placed, stored and/or transported, and accessed to remove thecontents. Typically such a container may be made of a material that iscompatible with the intended use and from which recovery of thecontained contents can be readily achieved. Preferred examples of suchcontainers include glass and/or plastic sealed or re-sealable tubes andampules, including those having a rubber septum or other sealing meansthat is compatible with withdrawal of the contents using a needle andsyringe. Such containers may, for instance, by made of glass or achemically compatible plastic or resin, which may be made of, or may becoated with, a material that permits efficient recovery of material fromthe container and/or protects the material from, e.g., degradativeconditions such as ultraviolet light or temperature extremes, or fromthe introduction of unwanted contaminants including microbialcontaminants. The containers are preferably sterile or sterilizable, andmade of materials that will be compatible with any carrier, excipient,solvent, vehicle or the like, such as may be used to suspend or dissolvethe herein described vaccine compositions and/or immunological adjuvantcompositions and/or antigens and/or recombinant expression constructs,etc.

Emulsion systems may also be used in formulating compositions of thepresent invention. For example, many single or multiphase emulsionsystems have been described. Oil in water emulsion adjuvants per se havebeen suggested to be useful as adjuvant composition (EP 0 399 843B),also combinations of oil in water emulsions and other active agents havebeen described as adjuvants for vaccines (WO 95/17210; WO 98/56414; WO99/12565; WO 99/11241). Other oil emulsion adjuvants have beendescribed, such as water in oil emulsions (U.S. Pat. No. 5,422,109; EP 0480 982 B2) and water in oil in water emulsions (U.S. Pat. No.5,424,067; EP 0 480 981 B). The oil emulsion adjuvants for use in thepresent invention may be natural or synthetic, and may be mineral ororganic. Examples of mineral and organic oils will be readily apparentto the man skilled in the art.

In a particular embodiment, a composition of the invention comprises anemulsion of oil in water wherein the GLA is incorporated in the oilphase. In another embodiment, a composition of the invention comprisesan emulsion of oil in water wherein the GLA is incorporated in the oilphase and wherein an additional component is present, such as aco-adjuvant, TLR agonist, or the like, as described herein.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system preferablycomprises a metabolizable oil. The meaning of the term metabolizable oilis well known in the art. Metabolizable can be defined as “being capableof being transformed by metabolism” (Dorland's illustrated MedicalDictionary, W. B. Saunders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts (such as peanut oil), seeds, and grains are commonsources of vegetable oils. Synthetic oils are also part of thisinvention and can include commercially available oils such as NEOBEE®and others.

Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene),for example, is an unsaturated oil which is found in large quantities inshark-liver oil, and in lower quantities in olive oil, wheat germ nil,rice bran oil, and yeast, and is a particularly preferred oil for use inthis invention. Squalene is a metabolizable oil virtue of the fact thatit is an intermediate in the biosynthesis of cholesterol (Merck index,10th Edition, entry no. 8619). Particularly preferred oil emulsions areoil in water emulsions, and in particular squalene in water emulsions.In addition, the most preferred oil emulsion adjuvants of the presentinvention comprise an antioxidant, which is preferably theoil.alpha.-tocopherol (vitamin E. EP 0 382 271 B1). WO 95/17210 and WO99/11241 disclose emulsion adjuvants based on squalene,alpha-tocopherol, and TWEEN® 80, optionally formulated with theimmunostimulants QS21 and/or 3D-MPL (which are discussed above). WO99/12565 discloses an improvement to these squalene emulsions with theaddition of a sterol into the oil phase. Additionally, a triglyceride,such as tricaprylin (C₂₇H₅₀O₆), may be added to the oil phase in orderto stabilize the emulsion (WO 98/56414).

The size of the oil droplets found within the stable oil in wateremulsion are preferably less than 1 micron, may be in the range ofsubstantially 30-600 nm, preferably substantially around 30-500 nm indiameter, and most preferably substantially 150-500 nm in diameter, andin particular about 150 nm in diameter as measured by photon correlationspectroscopy. In this regard, 80% of the oil droplets by number shouldbe within the preferred ranges, more preferably more than 90% and mostpreferably more than 95% of the oil droplets by number are within thedefined size ranges The amounts of the components present in the oilemulsions of the present invention are conventionally in the range offrom 2 to 10% oil, such as squalene; and when present, from 2 to 10%alpha tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylenesorbitan monooleate. Preferably the ratio of oil:alpha tocopherol isequal or less than 1 as this provides a more stable emulsion. Span 85may also be present at a level of about 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabilizer.

The method of producing oil in water emulsions is well known to theperson skilled in the art. Commonly, the method comprises the mixing theoil phase with a surfactant such as a PBS/TWEEN80® solution, followed byhomogenization using a homogenizer. For instance, a method thatcomprises passing the mixture once, twice or more times through asyringe needle would be suitable for homogenizing small volumes ofliquid. Equally, the emulsification process in a microfluidizer (M110Smicrofluidics machine, maximum of 50 passes, for a period of 2 minutesat maximum pressure input of 6 bar (output pressure of about 850 bar))could be adapted to produce smaller or larger volumes of emulsion. Thisadaptation could be achieved by routine experimentation comprising themeasurement of the resultant emulsion until a preparation was achievedwith oil droplets of the required diameter.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 2-AZIDO-2-DEOXY-D-GLUCOPYRANOSIDE (2)

Sodium azide (2.78 g, 42.7 mmol) was dissolved in water (7 mL) andtoluene (7 mL). The mixture was cooled to 0° C. under vigorous stirring.Triflic anhydride (4.57 mL, 27.2 mmol) was added dropwise, and themixture was stirred for 30 min at 0° C. The temperature was raised to10° C., and the biphasic mixture was stirred for 2 h. A saturatedaqueous solution of sodium hydrogencarbonate was added dropwise untilgas evolution had ceased. The two phases were separated, and the aqueouslayer was extracted with toluene (2×7 mL). The combined organic layerswere used in the subsequent diazo transfer reaction.

Glucose amine 1 (2.04 g, 9.45 mmol), sodium hydrogencarbonate (3.21 g,38.22 mmol), and copper(II) sulfate pentahydrate (90.5 mg, 0.362 mmol)were dissolved in water (12.3 mL). The triflic azide stock solutionprepared above (21 mL) was added, followed by the addition of methanol(81 mL) to yield a homogeneous system. The blue mixture was stirredvigorously at room temperature. Complete consumption of the amine wasmonitored by TLC (ninhydrin stain) and is also indicated by a colorchange of the mixture from blue to green. The solvents were removed invacuo with a rotary evaporator keeping the temperature strictly below25° C. The residue was purified by chromatography on silica gel (120 gRediSep column, eluting with a gradient of 0% through 40%methanol/dichloromethane over 50 min, 85 mL/min) to give product 2 (1.93g, 99%) as a colorless liquid. ¹H NMR (300 MHz, CD₃OD) (mixture ofdiastereomers 1/1) δ 5.18 (d, J=3.4 Hz, 0.5H), 4.51 (d, J=8.0 Hz, 0.5H),3.89-3.63 (m, 3H), 3.32-3.26 (m, 2H), 3.11-3.06 (m, 1H).

Example 2 2-AZIDO-2-DEOXY-4,6-O-BENZYLIDENE-D-GLUCOPYRANOSIDE (3)

To a solution of compound 2 (2.00 g, 9.75 mmol) in DMF (40 mL) was addedbenzaldehyde dimethyl acetal (1.65 g, 10.8 mmol) and camphorsulfonicacid (90 mg). The flask was connected to a vacuum system, and themixture was heated at 50° C. in an oil bath. After 3 h, the mixture wasconcentrated using a rotary evaporator. The residue was re-dissolved indiethyl ether (50 mL) and Et₃N (2 mL) followed by saturated sodiumbicarbonate (50 mL). The aqueous layer was extracted with diethyl ether(3×50 mL). The combined organic extracts were dried over sodium sulfateand filtered. After the removal of solvents using a rotary evaporator,the residue was purified by chromatography on silica gel (120 g RediSepcolumn, eluting with a gradient of 0% through 100% ethyl acetate/hexanesover 50 min, 85 mL/min) to give product 3 (2.58 g, 90%) as a colorlessliquid. ¹H NMR (300 MHz, CD₃OD) δ 7.49-7.32 (m, 5H), 5.58 (s, 1H), 4.64(d, J=3.8 Hz, 1H), 4.25-341 (m, 5H), 3.23-3.20 (m, 1H).

Example 3TERT-BUTYLDIMETHYLSILYL-2-AZIDO-4,6-O-BENZYLIDENE-2-DEOXY-β-D-GLUCOPYRANOSIDE(4)

t-Butyldimethylsilyl chloride (820 mg, 5.44 mmol) was added to a mixtureof compound 3 (1.45 g, 4.94 mmol) and imidazole (768 mg, 11.3 mmol) inCH₂Cl₂ (40 mL) at 0° C. After the solution was stirred overnight,saturated sodium bicarbonate (20 mL) was added, and the mixture wasextracted with diethyl ether (3×30 mL). The combined organic layers weredried over Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash column chromatography (80 g RediSep column, elutingwith a gradient of 0% through 70% ethyl acetate/hexanes over 40 min, 60mL/min) to yield product 4 (1.5 g, 74%) as a colorless solid. ¹H NMR(300 MHz, CDCl₃) δ 7.46-7.43 (m, 2H), 7.35-7.32 (m, 3H), 5.48 (s. 1H),4.59 (d, J=7.6 Hz, 1H), 4.23 (dd, J=10.2, 5.0 Hz, 1H), 3.73 (t, J=10.2Hz, 1H), 3.56-3.51 (m, 2H), 3.31-3.28 (m, 2H), 2.72 (d, J=2.2 Hz, 1H),0.91 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H).

Example 4TERT-BUTYLDIMETHYLSILYL-3-O-ALLYLOXYCARBONYL-2-AZIDO-4,6-O-BENZYLDIDINE-2-DEOXY-D-GLUCOPYRANOSIDE(5)

To a solution of compound 4 (1.50 g, 3.68 mmol) andtetramethylethylenediamine (TMEDA) (0.78 mL, 5.2 mmol) indichloromethane (DCM) (50 mL) at 0° C. was added allyl chloroformate(0.78 mL, 7.3 mmol) dropwise. The mixture was allowed to warm to roomtemperature, and the mixture was stirred at room temperature for 10 h.The mixture was diluted with DCM (50 mL) and washed with saturatedaqueous NaHCO₃ (2×100 mL) and brine (2×50 mL). The combined organiclayers were dried over Na₂SO₄, filtered, and concentrated in vacuo. Theresidue was purified by flash column chromatography (80 g RediSepcolumn, eluting with a gradient of 0% through 50% ethyl acetate/hexanesover 40 min, 60 mL/min) to yield product 5 (1.57 g, 87%) as a colorlesssolid. R_(f)=0.40 (hexanes/ethyl acetate, 3/1, v/v). ¹H NMR (300 MHz,CDCl₃) δ 7.44-7.41 (m, 2H), 7.35-7.32 (m, 3H), 5.98-5.85 (m, 1H), 5.48(s, 1H), 5.38-5.22 (m, 2H), 4.88 (t, J=11.4 Hz, 1H), 4.72-4.64 (m, 3H),4.32-4.27 (m, 1H), 3.81-3.65 (m, 2H), 3.50-3.42 (m, 2H), 0.94 (s, 9H),0.18 (s, 3H), 0.17 (s, 3H).

Example 5TERT-BUTYLDIMETHYLSILYL-3-O-ALLYLOXYCARBONYL-2-AZIDO-6-O-BENZYL-2-DEOXY-D-GLUCOPYRANOSIDE(6)

A suspension of compound 5 (320 mg, 0.651 mmol) and molecular sieves (4Å, 200 mg) in THF (5 mL) was stirred at room temperature for 1 h, andthen NaCNBH₃ (246 mg, 3.91 mmol) was added. A solution of hydrogenchloride (2 M in diethyl ether) was added dropwise to this mixture untilthe mixture became acidic (˜5 mL, pH=5). After being stirred another 0.5h, the reaction mixture was quenched with solid NaHCO₃, diluted withdiethyl ether (100 mL), and washed with saturated aqueous NaHCO₃ (2×100mL) and brine (2×50 mL). The organic layer was dried over Na₂SO₄,filtered, concentrated in vacuo, and the residue was purified by flashcolumn chromatography (40 g RediSep column, eluting with a gradient of0% through 100% ethyl acetate/hexanes over 40 min, 40 mL/min) to yieldproduct 6 (273 mg, 85%) as a colorless solid. R_(f)=0.42 (hexanes/ethylacetate, 4/1, v/v). ¹H NMR (300 MHz, CDCl₃) δ 7.39-7.34 (m, 5H),5.99-5.89 (m, 1H), 5.40-5.26 (m, 2H), 4.67-4.56 (m, 5H), 3.72-3.70 (m,3H), 3.48-3.46 (m, 2H), 3.37 (dd, J=9.6, 8.4 Hz, 1H), 3.01 (broad s,1H), 0.94 (s, 9H), 0.17 (s, 6H).

Example 6TERT-BUTYLDIMETHYLSILYL-3-O-ALLYLOXYCARBONYL-2-AZIDO-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-D-GLUCOPYRANOSIDE(7)

To a solution of compound 6 (5.47 g, 11.1 mmol) and 1H-tetrazole (3 wt %in acetonitrile, 35.5 mmol, 104 mL) was addedN,N-diethyl-1,5-dihydro-3H-2,4,3-benzodioxaphosphepin-3-amine (5.3 g. 22mmol). After the reaction mixture was stirred at room temperature for 15min, it was cooled to −20° C., stirred for another 10 min at thattemperature, and then mCPBA (8.40 g, 50-55 wt %, 24.4 mmol) was added.The reaction mixture was stirred at −20° C. for 20 min, and concentratedin vacuo. The residue was redissolved in DCM (30 mL) and washed withsaturated aqueous NaHCO₃ (40 mL). The aqueous layer was extracted withDCM (3×50 mL). The combined organic layers were dried over Na₂SO₄,filtered, and concentrated in vacuo. The residue was purified by flashcolumn chromatography (120 g RediSep column, eluting with a gradient of0% through 100% ethyl acetate/hexanes over 60 min, 85 mL/min) to yieldproduct 7 (4.85 g, 65%) as a pale yellow oil. R_(f)=0.40 (hexanes/ethylacetate, 1/1, v/v). ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.18 (m, 9H),5.98-5.85 (m, 1H), 5.41-5.05 (m, 6H), 4.64 (t, J=10.1 Hz, 1H), 4.58-4.52(m, 6H), 3.83 (d, J=9.0 Hz, 1H), 3.72-3.61 (m, 2H), 3.41 (dd, J=10.5,7.4 Hz, 1H), 0.92 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H).

Example 7TERT-BUTYLDIMETHYLSILYL-3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-(9-FLUORENYLMETHOXYCARBONYLAMINO)-D-GLUCOPYRANOSIDE(8)

Acetic acid (0.30 mL, 5.2 mmol) was added dropwise to a stirredsuspension of 7 (700 mg, 1.04 mmol) and zinc powder (676 mg, 10.4 mmol)in DCM (15 mL). The reaction mixture was stirred at room temperature for4 h, after which it was diluted with ethyl acetate (50 mL). The solidswere removed by filtration and washed with ethyl acetate (2×10 mL). Thecombined filtrates were washed with saturated aqueous NaHCO₃ (2×40 mL)and brine (2×40 mL). The organic phase was dried (MgSO₄) and filtered,and the filtrate was concentrated in vacuo to afford the crudeintermediate amine as a pale yellow oil. R_(f)=0.21 (hexanes/ethylacetate, 1/1, v/v). 9-Fluorenylmethyloxycarbonyl chloride (Fmoc-CI) (323mg, 1.25 mmol) was added to a stirred solution of the crude amine anddiisopropylethylamine (DIPEA) (0.22 mL, 1.3 mmol) in DCM (15 mL) at 0°C. The reaction mixture was warmed and stirred at room temperature for 5h, after which it was diluted with DCM (40 mL) and washed with brine(2×50 mL). The organic phase was dried (MgSO₄) and filtered. Thefiltrate was concentrated in vacuo. The residue was purified by silicagel column chromatography (40 g RediSep column, eluting with a gradientof 0% through 100% ethyl acetate/hexanes over 30 min, 40 mL/min) to giveproduct 8 (337 mg, 73% over two steps) as a white solid. R_(f)=0.54(hexanes/ethyl acetate, ill, v/v). ¹H NMR (300 MHz, CDCl₃) δ 7.78-7.20(m, 17H), 5.92-5.82 (m, 1H), 5.49-5.16 (m, 8H), 4.69-4.06 (m, 5H),4.49-4.28 (m, 2H), 3.88-3.61 (m, 3H), 3.60-3.51 (m, 2H), 3.32 (broad s,1H), 0.94 (s, 9H), 0.14 (s, 3H), 0.10 (s, 3H).

Example 83-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-(9-FLUORENYLMETHOXYCARBONYLAMINO)-D-GLUCOPYRANOSIDE(9)

Hydrogen fluoride/pyridine (6 mL, 0.2 mol) was added dropwise to astirred solution of 8 (6.00 g, 6.88 mmol) in THF (50 mL). The reactionmixture was stirred at room temperature for 12 h, after which it wasdiluted with diethyl ether (100 mL), and then washed with saturatedaqueous NaHCO₃ (2×40 mL) and brine (2×40 mL). The organic phase wasdried (MgSO₄) and filtered. The filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (120 g RediSepcolumn, eluting with a gradient of 0% through 80% ethyl acetate/hexanesover 60 min, 85 mL./min) to give product 9 (4.34 g, 83%) as a paleyellow oil. ¹H NMR (300 MHz, CDCl₃) δ 7.75-7.20 (m, 17H), 5.92-5.82 (m,1H), 5.27-5.06 (m, 9H), 4.59-4.55 (m, 5H), 4.41-4.39 (m, 1H), 4.25-4.01(m, 5H), 3.85-3.65 (m, 2H).

Example 9TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-DODECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(11)

A suspension of 10 (see preparation below) (350 mg, 0.172 mmol), zinc(1.3 g, 21 mmol), and acetic acid (0.70 mL, 12 mmol) in DCM (20 mL) wasstirred at room temperature for 12 h. The mixture was diluted withdiethyl ether. The solids were removed by filtration, and the residuewas washed with diethyl ether (2×10 mL). The combined filtrates werewashed with saturated aqueous NaHCO₃ (2×15 mL) and brine (2×15 mL). Theorganic phase was dried (MgSO₄) and filtered. The filtrate wasconcentrated in vacuo, and the residue was purified by silica gel columnchromatography (12 g RediSep column, eluting with a gradient of 0%through 60% ethyl acetate/hexanes over 35 min, 30 mL/min) to affordproduct 11 (220 mg, 64%) as a pale yellow syrup. R_(f)=0.29(hexanes/ethyl acetate, 5/2, v/v). ¹H NMR (300 MHz, CDCl₃) δ 7.37-7.24(m, 20H), 6.20 (d, J=7.2 Hz, 1H), 5.59 (t, J=9.6 Hz, 1H), 5.31 (m, 1H),5.12-4.97 (m, 6H), 4.62-4.44 (m, 7H), 4.05-3.24 (m, 9H), 2.68-2.12 (m,9H), 1.64-1.59 (m, 13H), 1.27 (broad m, 95H), 0.94 (m, 25H), 0.13 (s,6H). HRMS (m/z) (pos) calcd for C₁₁₇H₁₉₃N₂O₂₀PSi, 2005.37; found,2006.3729 [M+H]⁺.

Example 10TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-DODECANOYL]-2-[(R)-3-4-METHOXYBENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(12)

To a solution of amine 11 (93 mg, 0.046 mmol) in DCM (10 mL) was addedpyridine (21 mg, 0.27 mmol), (R)-3-(4-methoxybenzyloxy)tetradecanoylchloride (see preparation below, compound 35) (40 mg, 0.12 mmol), and4-dimethylaminopyridine (DMAP) (1 mg) at room temperature, and themixture was stirred overnight. The mixture was transferred to aseparatory funnel and diluted with diethyl ether (20 mL) and saturatedsodium bicarbonate (20 mL). The aqueous layer was extracted with diethylether (3×20 mL). The combined organic extracts were dried over sodiumsulfate, filtered and concentrated under reduced pressure. The residuewas purified by chromatography on silica gel (12 g RediSep column,eluting with a gradient of 0% through 80% ethyl acetate/hexanes over 35min, 30 mL/min) to give the product 12 (81 mg, 74%) as a colorlessliquid. R_(f)=0.34 (hexanes/ethyl acetate, 3/2, v/v). ¹H NMR (300 MHz,CDCl₃) δ 7.34-7.20 (m, 20H), 6.89-6.86 (m, 4H), 6.15 (t, J=9.0 Hz, 1H),5.57-5.55 (m, 1H), 5.31-4.99 (m, 8H), 4.57-4.44 (m, 11H), 4.06-3.33 (m,15H), 2.63-2.57 (m, 5H), 2.33-2.27 (m, 9H), 1.57 (m, 8H), 1.27 (broad m,112H), 0.88-0.82 (m, 27H), 0.08 (s, 3H), 0.04 (s, 3H). HRMS (m/z) (pos)calcd for C₁₃₉H₂₂₇N₂O₂₃PSi, 2351.62; found, 2352.6343 [M+H]⁺.

Example 11 Lipid A (13a)

A suspension of 12 (10 mg, 0.0042 mmol) and Pd-black (15.0 mg) inanhydrous THF (5 mL) was shaken under an atmosphere of H₂ (50 psi) atroom temperature for 30 h. The catalyst was removed by filtration. Theresidue was washed with THF (2×1 mL). The solution was cooled to −40° C.and neutralized with ammonia in methanol (0.1 mL, 7 M) and concentratedwithout heating in vacuo. The residue was purified by chromatography (12g RediSep column, eluting with chloroform/methanol/water 8/2/0.1 for 30min, 30 mL/min) to afford 13a (4 mg, 54%) as a colorless film. Theproduct was re-dissolved in water and methanol (v/v, 1/1, 2 mL) andlyophilized to obtain the product 13a as a white powder. ¹H NMR (500MHz, CDCl₃) δ 6.00-5.00 (m, 1H), 4.50-3.50 (m, 2H), 3.00-2.00 (m, 3H),2.00-1.00 (m, 50H), 0.81 (m, 18H). MS (Multimode, neg) calcd forC₉₆H₁₈₁N₂O₂₂P, 1745.28; found, 1745.0 [M−H]⁻.

Example 12 Lipid A (13b)

A suspension of 12 (27 mg, 0.011 mmol) and Pd-black (41.0 mg) inanhydrous THF (12 mL) was shaken under an atmosphere of H₂ (50 psi) atroom temperature for 30 h. The catalyst was removed by filtration. Theresidue was washed with THF (2×3 mL). The solution was neutralized withtriethylamine (TEA) (0.1 mL) and concentrated without heating in vacuo.The combined filtrates were concentrated in vacuo and purified bychromatography on silica (12 g RediSep column, eluting withchloroform/methanol/water 8/2/0.1 30 min, 30 mL/min) to afford 13b (5mg, 25%) as a colorless film. The product was re-dissolved in water andmethanol (v/v. 1/1, 2 mL) and lyophilized to obtain the product 13b as awhite powder. ¹H NMR (500 MHz, CDCl₃) δ 5.17 (broad, 2H), 4.23-3.62 (m,5H), 3.11-3.07 (q, J=2.8 Hz, 2H), 2.51-2.12 (m, 6H), 1.56-1.00 (m, 69H),0.92-0.84 (m, 18H). MS (Multimode, neg) calcd for C₉₆H₁₈₁N₂O₂₂P,1745.28; found, 1744.1 [M−H]⁻.

Example 13TERT-BUTYLDIMETHYLSILYL-6-O-[3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-(9-FLUORENYLMETHOXYCARBONYLAMINO)-β-D-GLUCOPYRANOSYL]-2-AZIDO-4-O-BENZYL-2-DEOXY-β-D-GLUCOPYRANOSIDE(15)

Compound 9 (89 mg, 0.12 mmol) was dissolved in anhydrous DCM (3 mL).Trichloroacetonitrile (1.0 mL) was added followed by sodium hydride (1.0mg, 60% in mineral oil). After 15 min, TLC indicated the presence of 9,so an additional quantity of sodium hydride (1 mg, 60% in mineral oil)was added. After 15 min, TLC indicated that the reaction was complete.The mixture was concentrated under vacuum and loaded onto a SiO₂ columnwhich was pretreated with Et₃N and eluted with 50% ethyl acetate/hexanesto provide the trichloroacetimidate intermediate (76.9 mg, 71%) whichwas used without further purification. A suspension oftrichloroacetimidate (76.9 mg, 0.0852 mmol), acceptor 14 (seepreparation below) (52.34 mg, 0.1277 mmol), and molecular sieves (4 Å,500 mg) in DCM (5.0 mL) was stirred at room temperature for 1 h. Themixture was cooled (−60° C.), and TMSOTf (1.54 μL, 0.0851 mmol) wasadded. After the reaction mixture was stirred for 30 min, it wasquenched with solid NaHCO₃. The solids were removed by filtration, andthe filtrate was concentrated in vacuo. The residue was purified bysilica gel column chromatography (hexanes/ethyl acetate, 2:1 (v/v)) togive 15 (55 mg, 40%) as a colorless solid. ¹H NMR (500 MHz, CD₃COCD₃) δ7.86-7.22 (m, 22H), 6.98 (d, J=9.0 Hz, 1H), 5.85 (m, 1H), 5.41 (t, J=9.0Hz, 1H), 5.38-5.21 (m, 3H), 5.10-5.02 (m, 3H), 4.91 (d, J=11.0 Hz, 2H),4.72-4.46 (m, 7H), 4.23-4.15 (m, 4H), 3.93-3.80 (m, 4H), 3.69-3.66 (m,1H), 3.54 (br s, 3H), 3.20 (dd, J₁=8.0 Hz, J₂=8.0 Hz, 1H), 0.95 (s, 9H),0.17 (s, 6H); ¹³C NMR (125 MHz, CD₃COCD₃) δ 207.00, 156.61, 155.51,145.22, 144.82, 142.06, 142.01, 139.98, 139.57, 136.68, 136.62, 133.02,132.94, 129.85, 129.83, 129.15, 129.05, 128.95, 128.91, 128.82, 128.61,128.49, 128.41, 128.21, 128.17, 128.0, 127.92, 126.19, 126.09, 125.98,120.79, 118.60, 118.52, 101.41, 97.57, 78.78, 78.10, 76.84, 75.98,75.88, 75.43, 75.30, 75.17, 74.70, 74.07, 70.63, 69.76, 69.64, 69.27,69.15, 69.10, 69.02, 68.97, 67.73, 67.17, 57.29, 54.94, 26.11, 18.51; HRMS (m/z) calcd for C₅₉H₆₉N₄O₁₆PSi [M+H]⁺, 1149.4293; found, 1149.4238.

Example 14TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-2-DEOXY-β-D-GLUCOPYRANOSIDE(16)

1,8-Diazabicylco[5.4.0]undec-7-ene (220 μL, 1.47 mmol) was addeddropwise to a solution of 15 (800 mg, 0.696 mmol) in DCM (10 mL). Thereaction mixture was stirred at room temperature for 1 h, after which itwas concentrated in vacuo. The residue was purified by silica gel columnchromatography (DCM/methanol, 100:1 through 100:3 (v/v)) to afford thefree amine (648 mg, 99%) as a colorless syrup. ¹H NMR (500 MHz, CDCl₃) δ7.36-7.17 (m, 14H), 5.96-5.88 (m, 1H), 5.40-5.06 (m, 7H), 4.84-4.50 (m,9H), 4.21 (d, J=13.5 Hz, 1H), 4.15-4.11 (m, 1H), 3.82 (m, 1H), 3.79-3.42(m, 5H), 3.34-3.19 (m, 2H), 2.96-2.90 (m, 1H), 2.34 (d, J=4.5 Hz, 1H),0.90 (s, 9H), 0.13 (s, 6H). HRMS (m/z) calcd for C₄₄H₅₉N₄O₁₄PSi [M+H]⁺,927.3613; found, 927.3569.

N,N-Dicyclohexylcarbodiimide (DCC) (230 mg, 1.11 mmol) was added to astirred solution of (R)-3-dodecanoyl-tetradecanoic acid (see preparationbelow, compound 40) (381 mg, 0.81 mmol) in DCM (10 mL). After thereaction mixture was stirred for 10 min, the free amine (648 mg, 0.699mmol) in DCM (10 mL) was added, and stirring was continued for another12 h. The insoluble materials were removed by filtration, and theresidue was washed with DCM (2×2 mL). The combined filtrates wereconcentrated in vacuo, and the residue was purified by silica gel columnchromatography (hexanes/ethyl acetate, 2:1 (v/v)) to give 16 (450 mg,47%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.35-7.17 (m, 14H),5.94-5.86 (m, 2H), 5.47 (t, J=9.0, 10.5 Hz, 1H), 5.37 (d, J=2.5 Hz, 1H),5.34 (d, J=2.5 Hz, 1H), 5.24 (d, J=13.5 Hz, 1H), 5.13-4.97 (m, 6H), 4.75(d, J=11.0 Hz, 1H), 4.66-4.49 (m, 7H), 4.00 (d, J=17.0 Hz, 2H), 3.83 (d,J=10.5 Hz, 1H), 3.75-3.56 (m, 4H), 3.49-3.36 (m, 5H), 3.20 (m, 1H),2.42-2.17 (m, 4H). 1.93 (d, J=11.5 Hz, 1H), 1.70 (m, 2H), 1.23 (br s,36H), 0.92 (s, 9H), 0.89-0.86 (m, 6H), 0.14 (s, 6H); HRMS (m/z) calcdfor C₇₂H₁₁₁N₄O₁₇PSi [M+H]⁺, 1363.7529; found. 1363.7487.

Example 15TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(17)

A mixture of (R)-3-benzyloxytetradecanoic acid (see preparation below,compound 33) (120 mg, 0.540 mmol) and DCC (171 mg, 0.830 mmol) in DCM (5mL) was stirred at room temperature for 10 min, and then disaccharide 16(451 mg, 0.331 mmol) in DCM (5 mL) and DMAP (25 mg, 0.21 mmol) wereadded. The reaction mixture was stirred at room temperature for 14 h,after which the solids were removed by filtration. The residue waswashed with DCM (2×4 mL). The combined filtrates were concentrated invacuo, and the residue was purified by silica gel column chromatography(hexanes/ethyl acetate, 4:1 (v/v)) to give 17 (540 mg, 97%) as a whitesolid. R_(f)=0.41 (hexanes/ethyl acetate, 2:1 (v/v)). ¹H NMR (500 MHz,CDCl₃) δ 7.33-7.15 (m, 19H), 5.94-5.85 (m, 2H), 5.47 (t, J=9.5 Hz, 1H),5.37 (d, J=17.5 Hz, 1H), 5.22 (d, J=10.0 Hz, 1H), 5.10-4.95 (m, 7H),4.62-4.43 (m, 10H), 4.0-3.96 (m, 3H), 3.90-3.81 (m, 2H), 3.74-3.67 (m,3H), 3.56-3.42 (m, 6H), 3.33-3.27 (m, 1H), 2.60-2.21 (m, 6H), 1.24 (brs, 54H), 0.91 (s, 9H), 0.87-0.84 (m, 9H), 0.14 (s. 6H). HRMS (m/z) calcdfor C₉₃H₁₄₃N₄O₁₉PSi [M+H]⁺, 1679.9931; found, 1679.9934.

Example 16TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(18)

Tetrakis(triphenylphosphine)palladium (228 mg, 0.198 mmol) was added toa solution of 17 (1.66 g, 0.980 mmol), n-BuNH₂ (0.19 mL, 1.97 mmol), andHCOOH (74.5 μL, 1.98 mmol) in THF (20 mL). After the reaction mixturewas stirred at room temperature for 20 min, it was diluted with DCM (40mL), and washed successively with water (40 mL), saturated aqueousNaHCO₃ (2×40 mL), and brine (40 mL). The organic phase was dried (MgSO₄)and filtered. The filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (hexanes/ethyl acetate, 4:3(v/v)) to give compound 18 (1.43 g, 91%). R_(f)=0.5 (hexanes/ethylacetate, 1:1 (v/v)). ¹H NMR (500 MHz, CDCl₃) δ 7.33-7.11 (m, 19H), 6.2(d, J=7.5 Hz, 1H), 5.46 (t, J=9.0 Hz, 1H), 5.04-4.90 (m, 9H), 4.55-4.38(m, 8H), 3.92 (d, J=10.0 Hz, 1H), 3.84-3.76 (m, 1H), 3.75-3.7 (m, 4H),3.53-3.44 (m, 2H), 3.43-3.32 (m, 2H), 3.25-3.20 (m, 1H), 2.61-2.10 (m,12H), 1.23 (br s, 54H), 0.90 (s, 9H), 0.88-0.84 (m, 9H), 0.12 (s, 6H).HRMS (m/z) calcd for C₈₉H₁₃₉N₄O₁₇PSi [M+H]⁺, 1595.972; found, 1595.9713.

Example 17TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-(P-METHOXY)BENZYLOXYTETRADECANOYL]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(19)

A solution of (R)-3-(p-methoxy)benzyloxy-tetradecanoic acid (seepreparation below, compound 34, 424 mg, 1.16 mmol) and DCC (369 mg, 1.79mmol) in DCM (15 mL) was stirred at room temperature for 10 min, and thealcohol 18 (1.43 g, 0.896 mmol) in DCM (10 mL) and DMAP (54.72 mg,0.4479 mmol) were added. The reaction mixture was stirred for another 14h, after which the solids were removed by filtration and washed with DCM(2×5 mL). The combined filtrates were concentrated in vacuo. The residuewas purified by silica gel column chromatography (hexanes/ethyl acetate,4:1 (v/v)) to afford 19 (1.15 g, 66%) as a white solid. R_(f)==0.46(hexanes/ethyl acetate, 2:1 (v/v)). ¹H NMR (500 MHz, CDCl₃) δ 7.38-6.79(m, 23H), 5.73 (d. J=8.0 Hz, 1H), 5.55 (t, J=9.5 Hz, 1H), 5.20-4.88 (m,8H), 4.66-4.47 (m, 12H), 4.33 (d, J=12.5 Hz, 1H), 4.0-3.66 (m, 12H),3.61-3.40 (m, 5H), 3.36-3.27 (m, 3H), 2.67 (d, J=6.0 Hz, 2H), 2.60-2.22(m, 6H), 1.27 (br s, 72H), 0.93 (s, 9H), 0.92-0.87 (m, 12H), 0.16 (s,6H). HRMS (m/z) calcd for C₁₁₁H₁₇₃N₄O₂₀PSi [M+H]⁺, 1942.2228; found,1942.2289.

Example 18TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-TETRADECANOYLOXY-TETRADECANOYL]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(10)

To a stirred solution of 19 (1.15 g, 0.592 mmol) in a mixture of DCM andH₂O (11 mL, 10:1 (v/v)) was added2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (202 mg, 0.890 mmol).The reaction mixture was stirred at room temperature for 1 h, afterwhich it was diluted with DCM. The mixture was washed with brine (20mL), dried (MgSO₄), and concentrated in vacuo. The residue was purifiedby silica gel column chromatography (hexanes/ethyl acetate, 3:1 (v/v))to give the alcohol as a colorless syrup (1.01 g, 94%). R_(f)=0.50(hexanes/ethyl acetate, 5:3 (v/v)). Myristoyl chloride (0.74 mL, 2.7mmol) was added to a solution of the alcohol (1.01 g, 0.554 mmol), andpyridine (0.35 mL, 4.33 mmol) in DCM (20 mL). After the reaction mixturewas stirred at room temperature for 12 h, it was diluted with DCM andwashed with saturated aqueous NaHCO₃ (2×40 mL) and brine (40 mL). Theorganic phase was dried (MgSO₄) and filtered. The filtrate wasconcentrated in vacuo. The residue was purified by silica gel columnchromatography (hexanes/ethyl acetate, 4:1 (v/v)) to afford 10 (680 mg,57%) as a white solid. R_(f)==0.46 (hexanes/ethyl acetate, 5:2 (v/v)).¹H NMR (500 MHz, CDCl₃) δ 7.37-7.24 (m, 19H), 6.23 (d, J=7.5 Hz, 1H),5.58 (t, =J₂=9.5 Hz, 1H), 5.32-5.27 (m, 1H), 5.16-4.99 (m, 6H),4.78-4.44 (m, 7H), 4.03 (d. J=10.5 Hz, 1H), 3.99-3.20 (m, 10H),2.65-2.21 (m, 10H), 1.61-1.51 (m, 10H), 1.27 (br s, 94H), 1.21 (br s,25H), 0.12 (s, 6H).

Example 19TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-2-DEOXY-β-D-GLUCOPYRANOSIDE(20)

Compound 15 (1.23 g, 1.07 mmol) was acylated in a manner similar to thesynthesis of compound 16 (Example 14) using (DCC, 430 mg, 2.08 mmol),required lipid (Compound 40, Example 36, 630 mg, 1.59 mmol), andtriethylamine (161 mg, 1.59 mmol) to provide 20 (1.05 g, 81%) as acolorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.35-7.17 (m, 14H), 5.91-5.86(m, 2H), 5.47 (t, J=9.0, 10.5 Hz, 1H), 5.34 (d, J=17 Hz, 1H), 5.24 (d,J=10.5 Hz, 1H), 5.10-4.98 (m, 8H), 4.75 (d, J=11.5 Hz, 1H), 4.66-4.49(m, 8H), 4.00 (d, J=11.0 Hz, 2H), 3.83 (d, J=11.0 Hz, 1H), 3.75-3.69 (m,2H), 3.49-3.36 (m, 4H), 3.20 (m, 1H), 2.40-2.26 (m, 4H), 1.24 (br s,32H), 0.92 (s, 9H), 0.89-0.86 (m, 6H), 0.14 (s, 6H); MS (Multimode, pos)m/z=1307 [M+H]⁺.

Example 20TERT-BUTYLDIMETHYLSILYL-6-O-{3-O-ALLYLOXYCARBONYL-6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(21)

Compound 20 (1.43 g, 1.18 mmol) was acylated in a manner similar to thesynthesis of compound 17 (Example 15) using (DCC, 453 mg, 2.20 mmol),required lipid (477 mg, 1.43 mmol), and N,N-dimethyl-4-aminopyridine (67mg, 0.548 mmol) to provide 21 (1.60 g, 83%) as a colorless oil. ¹H NMR(500 MHz, CDCl₃) δ 7.33-7.15 (m, 19H), 5.94-5.85 (m, 2H), 5.48 (t, J=9.0Hz, 1H), 5.34 (d, J=17.5 Hz, 1H), 5.22 (d, J=10.0 Hz, 1H), 5.12-4.96 (m,7H), 4.63-4.46 (m, 11H), 3.97 (d, J=10.5 Hz, 1H), 3.89-3.85 (m, 2H),3.74-3.68 (m, 3H), 3.55-3.52 (m, 2H), 3.47-3.41 (m, 1H), 3.28 (m, 1H),2.61-2.22 (m, 8H), 1.59-1.52 (m, 6H), 1.98 (m, 2H), 1.23 (br s, 44H),0.90 (s, 9H), 0.88-0.84 (m, 9H), 0.12 (s, 6H); MS (Multimode, pos)m/z=1625 [M+H]⁺.

Example 21TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DODECANOYLOXY-TETRADECANOYLAMINO]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(22)

Compound 21 (1.60 g, 0.985 mmol) was reacted in a manner analagous tothe synthesis of compound 18 (Example 16). Accordingly,tetrakis(triphenylphosphine)palladium, (227 mg, 0.196 mmol), formic acid(74 μL, 1.97 mmol), and n-butylamine (144 mg, 1.97 mmol) to provide 22(1.25 g, 82%) as a yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 7.33-7.15 (m,19H), 6.20 (d, J=7.5 Hz, 1H), 5.38-4.95 (m, 6H), 4.86 (d, J=8.0 Hz, 1H),4.60-4.46 (m, 10H), 3.97-3.71 (m, 8H), 3.68-3.48 (m, 5H), 3.31-3.27 (m,3H), 2.62-2.55 (m, 2H), 2.50-2.42 (m, 3H), 2.40-2.22 (m, 5H), 1.23 (brs, 44H), 0.90 (s, 9H), 0.88-0.84 (m, 9H), 0.12 (s, 6H); MS (Multimode,pos) m/z=1539 [M+H]⁺.

Example 22TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-(P-METHOXY)BENZYLOXYTETRADECANOYL]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(23)

Compound 22 (1.25 g, 0.811 mmol) was acylated in a manner similar to thesynthesis of compound 19 (Example 17) using (DCC, 335 mg, 1.62 mmol),required lipid (Compound 34, Example 32, 386 mg, 1.06 mmol), andN,N-dimethyl-4-aminopyridine (50 mg, 0.41 mmol) to provide 23 (440 mg,29%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.38-6.79 (m, 23H),5.71 (d, J=7.5 Hz, 1H), 5.55 (t, J=9.5 Hz, 1H), 5.06-4.85 (m, 9H),4.66-4.45 (m, 12H), 3.97 (d, J=11.0 Hz, 1H), 3.90-3.69 (m, 9H),3.60-3.55 (m, 3H), 3.37-3.29 (m, 2H), 2.65 (d, J=7.5 Hz, 2H), 2.61-2.55(m, 1H), 2.48-2.42 (m, 1H), 2.35-2.21 (m, 3H), 2.11-2.05 (m, 1H),1.62-1.59 (m, 8H), 1.27 (br s, 62H), 0.93 (s, 9H), 0.92-0.87 (m, 12H),0.16 (s, 6H); MS (Multimode, pos) m/z=1886[M+H].

Example 23TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-DECANOYLOXY-TETRADECANOYL]-β-D-GLUCOPYRANOSYL}-2-AZIDO-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-DEOXY-β-D-GLUCOPYRANOSIDE(24)

Compound 23 (446 mg, 0.236 mmol) was first deprotected using DDQ (80 mg,0.35 mmol) following the procedure for intermediate 10 for Target A.This intermediate (343 mg, 0.194 mmol) was then acylated in a mannersimilar to the synthesis of compound 10 for Target A using decanoylchloride (185 mg, 0.970 mmol) and pyridine (123 mg, 1.55 mmol) toprovide 24 (343 mg, 76%) as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ7.39-7.22 (m, 14H), 6.15 (d, J=7.5 Hz, 1H), 5.54 (t, J=9.5 Hz, 1H),5.28-5.24 (m, 1H), 5.14-4.96 (m, 8H), 4.60-4.45 (m, 10H), 3.99 (d,J=10.5 Hz, 1H), 3.90-3.85 (m, 1H), 3.80-3.65 (m, 4H), 3.55 (m, 3H),3.46-3.39 (m, 1H), 3.32-3.27 (m, 1H), 2.66-2.53 (m, 3H), 2.46-2.41 (m,1H), 2.35-2.18 (m, 7H), 1.61-1.51 (m, 10H), 1.26 (br s, 78H), 0.95 (s,9H), 0.92-0.90 (m, 15H), 0.19 (s, 3H), 0.18 (s, 3H).

Example 24TERT-BUTYLDIMETHYLSILYL-6-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-DECANOYLOXY-TETRADECANOYL]-β-D-GLUCOPYRANOSYL}-4-O-BENZYL-3-O—[(R)-3-BENZYLOXYTETRADECANOYL]-2-[(R)-3-BENZYLOXY-TETRADECANOYLAMINO]-2-DEOXY-β-D-GLUCOPYRANOSIDE(25)

A suspension of 24 (296 mg, 0.154 mmol), zinc (100 mg, 1.52 mmol), andacetic acid (53 μL, 0.93 mmol) in DCM (10 mL) was stirred at roomtemperature for 12 h, after which it was diluted with ethyl acetate (25mL). The solids were removed by filtration and washed with ethyl acetate(2×25 mL), and the combined filtrates were washed with saturated aqueousNaHCO₃ (2×100 mL) and brine (200 mL). The organic phase was dried(Na₂SO₄) and filtered. The filtrate was concentrated in vacuo. Theresidue was purified by silica gel column chromatography (hexanes/ethylacetate, 2.5:1 (v/v)) to afford the amine as a pale yellow syrup (245mg, 84%). ¹H NMR (500 MHz, CDCl₃) δ 7.39-7.22 (m, 14H), 6.15 (d, J=7.5Hz, 1H), 5.54 (t, J=9.5 Hz, 1H), 5.29-5.23 (m, 1H), 5.13-4.93 (m, 8H),4.62-4.30 (m, 9H), 4.00 (d, J=10.5 Hz, 1H), 3.88-3.65 (m, 6H), 3.56-3.53(m, 2H), 3.46-3.41 (m, 1H), 2.66-2.58 (m, 4H), 2.54-2.45 (m, 2H),2.35-2.17 (m, 7H), 1.64-1.42 (m, 12H), 1.26 (br s, 78H), 0.87 (s, 24H),0.13 (s, 6H).

The amine was added to a stirred solution of(R)-3-benzyloxytetradecanoyl chloride (228 mg, 0.646 mmol), DMAP (15.79mg, 0.1292 mmol), and pyridine (83 μL, 1.0 mmol) in DCM (5.0 mL). Thereaction mixture was stirred for 14 h. The mixture was diluted withCH₂Cl₂ and was washed with saturated NaHCO₃/brine dried under Na₂SO₄ andconcentrated under vacuum. The residue was purified by silica gel TLCchromatography (hexanes/ethyl acetate, 3.5:1 (v/v)) to give 25 (450mg, >100%) as a white solid. R_(f)=0.54 (hexanes/ethyl acetate, 2:1(v/v)). ¹H NMR (500 MHz, CDCl₃) δ 7.39-7.22 (m, 19H), 6.14-6.10 (m, 2H),5.57 (t, J=9.5 Hz, 1H), 5.29-5.24 (m, 1H), 5.13-4.93 (m, 7H), 4.61-4.41(m, 10H), 4.00 (d, J=10.5 Hz, 1H), 3.89-3.79 (m, 8H), 3.72-3.66 (m, 4H),3.57-3.35 (m, 3H), 2.73-2.57 (m, 10H), 2.39-2.15 (m, 10H), 1.71-1.64 (m,7H), 1.26 (br s, 93H), 0.88 (s, 24H), 0.83 (s, 9H).

Example 256-O-{6-O-BENZYL-2-DEOXY-4-O-(1,5-DIHYDRO-3-OXO-3λ⁵-3H-2,4,3-BENZODIOXAPHOSPHEPIN-3-YL)-2-[(R)-3-DECANOYLOXY-TETRADECANOYLAMINO]-3-O—[(R)-3-DECANOYLOXY-TETRADECANOYL]-β-D-GLUCOPYRANOSYL}-4-O-BENZYL-3-O—[(R)-3-BENZYLOXY-TETRADECANOYL]-2-[(R)-3-BENZYLOXY-TETRADECANOYLAMINO]-2-DEOXY-α-D-GLUCOPYRANOSE(26)

Hydrogen fluoride/pyridine (1.12 mL, 43.1 mmol) was added dropwise to astirred solution of 25 (450 mg, 0.204 mmol) in THF (5 mL). The reactionmixture was stirred at room temperature for 14 h. The mixture wasdiluted with ethyl acetate (100 mL) and washed with saturated aqueousNaHCO₃ (2×80 mL) and brine. The organic phase was dried (Na₂SO₄) andfiltered. The filtrate was concentrated in vacuo. The residue waspurified by silica gel column chromatography (hexanes/ethyl acetate, 3:1through 4:3 (v/v)) to give 26 (180 mg, 42%) as a white solid. ¹H NMR(500 MHz, CDCl₃) δ 7.39-7.19 (m, 19H), 6.31 (d, J=7.0 Hz, 1H), 6.24 (d,J=9.5 Hz, 1H), 5.57-5.48 (m, 2H), 5.40 (t, J=9.5 Hz, 1H), 5.28-5.21 (m,1H), 5.14-4.96 (m, 8H), 4.68-4.41 (m, 12H), 4.23-4.19 (m, 1H), 4.13-4.06(m, 1H), 3.94-3.66 (m, 9H), 3.38-3.28 (m, 2H), 2.67-2.58 (m, 3H),2.44-2.20 (m, 11H), 1.58 (br s, 12H), 1.26 (br s, 93H), 0.91-0.81 (m,18H).

Example 26(3R)-((2R,3S,4R,5S)-3-((R)-3-(DECANOYLOXY)TETRADECANAMIDO)-2-(((3S,4R,5S)-3,6-DIHYDROXY-5-((R)-3-HYDROXYTETRADECANAMIDO)-4-((R)-3-HYDROXYTETRADECANOYLOXY)TETRAHYDRO-2H-PYRAN-2-YL)METHOXY)-6-(HYDROXYMETHYL)-5-(PHOSPHONOOXY)TETRAHYDRO-2H-PYRAN-4-YL)3-(DECANOYLOXY)TETRADECANOATE (IX)

Compound 26 (180 mg, 0.0858 mmol) was dissolved in anhydrous THF (15mL). Palladium black (0.225 g) was added to the mixture and washydrogenated under 50 psi hydrogen atmosphere overnight. The mixture wasfiltered through a bed of diatomaceous earth. The filtrate was cooled to−40° C. and a solution of ammonia in methanol (1.8 mL, 4 M) was added.The mixture was concentrated under vacuum without heating. The residuewas purified by silica gel chromatography eluting with a mixture ofchloroform/methanol/water, 80:20:1 (v/v) to give the desired compound(IX) (102 mg, 73%). Analysis by TLC and ¹H NMR showed the presence ofgrease and a faint close running spot (TLC in CH₂Cl₂/CMA, 4:1). Theresidue was subjected to chromatography (12 g RediSep column, elutedwith a gradient of isocratic CH₂Cl₂ for 5 column volumes (CVs), agradient through 25% CMA over 10 CVs, isocratic for 10 CVs, a gradientthough 100% CMA over 10 CVs, isocratic at 100% CMA for 10 CVs, 20mL/min) to give the desired product (57 mg, 25%). TLC analysis of thecombined and concentrated fractions still indicated a very small amountof impurity running just above the desired product. The residue wasre-purified by silica gel chromatography (two 12 g RediSep columns inseries, same gradient as above) to provide 8.9 mg of the desired productpure by TLC and 11.9 mg of slightly impure product after dissolving inmethanol/water/chloroform and freeze-drying. Total yield (20.8 mg, 14%)as an off white solid. R_(f)=0.40 CMA. ¹H NMR (500 MHz, CDCl₃) δ5.40-5.30 (br s, 2H), 4.10-4.00 (m, 4H), 3.70-3.60 (m, 4H), 2.83-2.76(m, 1H), 2.75-2.20 (m, 13H), 2.10-1.90 (broad, 9H), 1.40-1.00 (broad,106H), 0.90-0.70 (broad, 18H). MS (Multimode, Neg) m/z=1632 [M−H]⁻.

Example 27 METHYL 3-OXOTETRADECANOATE (0.29)

To a suspension of magnesium ethoxide (10.82 g, 94.61 mmol) in1,4-dioxane (100 mL) was added methyl hydrogen malonate (25.0 g, 189mmol) in 1,4-dioxane (100 mL). The resulting slurry was stirredovernight. The mixture was concentrated in vacuo. In a separate flask,lauric acid (28, 20.85 g, 104.1 mmol) was dissolved in 1,4-dioxane (50mL) and a solution of CDI (16.88 g, 104.1 mmol) in 1,4-dioxane (150 mL)was added at room temperature. The resulting solution was stirredovernight. The mixture was then transferred to the methyl magnesiummalonate flask. The resulting suspension was refluxed overnight. Themixture was concentrated in vacuo. The residue was redissolved in DCM(300 mL) and filtered through a silica plug (10 g). The solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography (360 g RediSep column, eluting with a gradientof 0% through 30% ethyl acetate/hexanes over 80 min, 100 mL/min) toafford product 29 (17 g, 61%) as a pale yellow syrup.

Example 28 (R)-METHYL 3-HYDROXYTETRADECANOATE (30)

A slurry of methyl 3-oxotetradecanoate (29, 29.0 g, 113 mmol) inmethanol (120 mL) was purged in a 300 mL high pressure reactor glasssleeve with N₂ for 10 minutes.Dichloro-R-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ruthenium (897mg, 1.10 mmol) was added. The mixture was placed in a Parr 5500 seriescompact reactor. The reactor was charged with H₂ (60 psi) and vented 3times. The reactor was charged with H₂ (60 psi) and stirred (1200 rpm)and heated to 50° C. for 20 h. The reactor was cooled to roomtemperature, and the resulting orange solution was concentrated invacuo. The residue was purified by silica gel chromatography (120 gRediSep column, eluting with a gradient of 0% through 40% ethylacetate/hexanes over 60 min, 85 ml/min) to provide product 30 (28.5 g,97% yield) as a white solid.

Example 29 (R)-METHYL 3-(BENZYLOXY)TETRADECANOATE (31)

To a solution of compound 30 (2.8 g, 10.83 mmol) and benzyltrichloroacetimidate (3.4 g, 14 mmol) in DCM (100 mL) was addedtrifluoromethanesulfonic acid (0.24 mL, 2.7 mmol) dropwise at 0° C. Theresulting mixture was stirred at 0° C. for 6 h and warmed to roomtemperature. The mixture was washed with a saturated solution of NaHCO₃(300 mL) and water (300 mL) and the organic layer dried over Na₂SO₄. Thedrying agent was removed by filtration, and the solvents removed using arotary evaporator. The residue was purified by chromatography on silicagel (80 g RediSep column, eluting with a gradient of 0% through 30%ethyl acetate/hexanes over 60 min, 60 mL/min) to give the product 31(1.2 g, 32%) as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 7.30-7.05(m, 5H), 4.51 (s, 2H), 3.90-3.80 (m, 1H), 3.70 (s, 3H), 2.58-2.45 (m,2H), 1.80-1.60 (m, 2H), 1.50-1.20 (m, 18H), 0.85 (t, J=5.8 Hz, 3H).

Example 30 (R)-3-(BENZYLOXY)TETRADECANOIC ACID (33)

Ester 31 (1.3 g, 3.73 mmol) was dissolved in THF/MeOH/CH₃CN mixture(v/v/v, 1/1/1, 90 mL). Lithium hydroxide monohydrate (235 mg, 5.6 mmol)as a solution in water (30 mL) was added, and the mixture stirredovernight. The solvent amount was reduced in vacuo to about 30 mL. Tothe remaining aqueous solution was added 1 M hydrochloric acid to bringthe pH down to 3. The aqueous layer was extracted with diethyl ether(3×40 mL). The combined organic extracts were dried over sodium sulfate.The drying agent was removed by filtration, and the solvents removedusing a rotary evaporator. The residue was purified by chromatography onsilica gel (40 g RediSep column, eluting with a gradient of 0% through50% ethyl acetate/hexanes over 40 min, 40 mL/min) to give the product 33(990 mg, 79%) as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 7.30-7.05(m, 5H), 4.51 (s, 2H), 3.90-3.80 (m, 1H), 2.58-2.45 (m, 2H), 1.80-1.60(m, 2H), 1.50-1.20 (m, 18H), 0.85 (t, J=5.8 Hz, 3H).

Example 31 (R)-METHYL 3-(4-METHOXYBENZYLOXY)TETRADECANOATE (32)

To a solution of compound 30 (3.50 g, 12.9 mmol) and 4-methoxybenzyltrichloroacetimidate (4.65 g, 17.3 mmol) in DCM (100 mL) was addedcamphorsulfonic acid (450 mg, 1.92 mmol). The mixture was stirredovernight at room temperature. The mixture was washed with a saturatedsolution of NaHCO₃ (300 mL) and water (300 mL) and dried over Na₂SO₄.The drying agent was removed by filtration and the solvents removedusing a rotary evaporator. The residue was purified by chromatography onsilica gel (120 g RediSep column, eluting with a gradient of 0% through30% ethyl acetate/hexanes over 70 min, 85 mL/min) to give the product 32(4.01 g, 81%) as a colorless liquid.

Example 32 (R)-3-(4-METHOXYBENZYLOXY)TETRADECANOIC ACID (34)

Ester 32 (4.01 g, 10.4 mmol) was dissolved in THF/MeOH/CH₃CN mixture(v/v/v, 1/1/1, 90 mL). Lithium hydroxide monohydrate (874 mg, 20.8 mmol)as a solution in water (30 mL) was added, and the mixture stirredovernight. The solvent amount was reduced in vacuo to about 30 mL. Tothe remaining aqueous solution was added hydrochloric acid (1 M) tobring the pH down to 3. The aqueous layer was extracted with diethylether (3×40 mL). The combined organic extracts were dried over sodiumsulfate. The drying agent was removed by filtration and the solventsremoved using a rotary evaporator. The residue was purified bychromatography on silica gel (120 g RediSep column, eluting with agradient of 0% through 50% ethyl acetate/hexanes over 60 min, 85 mL/min)to give the product 34 (3.37 g, 89%) as a colorless liquid. ¹H NMR (300MHz, CDCl₃) 7.22 (d, J=6.1 Hz, 2H), 6.82 (d, J=6.1 Hz, 2H), 4.46 (s,2H), 3.81 (m, 1H), 3.75 (s, 3H), 2.65-2.49 (m, 2H), 1.80-1.60 (m, 2H),1.50-1.20 (m, 18H), 0.85 (t, J=5.8 Hz, 3H).

Example 33 (R)-3-(4-METHOXYBENZYLOXY)TETRADECANOYL CHLORIDE (35)

To a solution of acid 34 (500 mg, 1.37 mmol) in DCM (5 mL) was addeddimethylformamide (DMF) (100 mg, 1.37 mmol), and the resulting mixturewas cooled to −10° C. Oxalyl chloride (174 mg, 1.37 mmol) in DCM (5 mL)was added dropwise. The solution was allowed to warm to room temperatureover 1 h. After TLC analysis showed no acid present, the mixture wasconcentrated in vacuo and used without further purification.

Example 34 (R)-2-OXO-2-PHENYLETHYL 3-HYDROXYTETRADECANOATE (37)

To a solution of (R)-3-hydroxytetradecanoic acid (36, see preparationbelow) (9.55 g, 39.1 mmol) and triethylamine (5.90 g, 58.6 mmol) inethyl acetate (500 mL) was added 2-bromoacetophenone (7.90 g, 39.1 mmol)at room temperature. The mixture was stirred at room temperature for 14h. The precipitate was removed by filtration, and the filtrate wasconcentrated in vacuo. The residue was purified by silica gelchromatography (120 g RediSep column, eluting with a gradient of 0%through 30% ethyl acetate/hexanes over 50 min, 85 mL/min) to give theproduct 37 (10.2 g, 72% yield) as a white solid.

Example 35 (R)-2-OXO-2-PHENYLETHYL-3-DECANOYLOXYTETRADECANOATE (39)

To a solution of 37 (4.80 g, 13.2 mmol) and pyridine (2.10 g, 26.5 mmol)in DCM (100 mL) at 0° C. was added decanoyl chloride (38, 2.8 g, 4.8mmol). The mixture was stirred for 14 h allowing the temperature of themixture to rise to room temperature. The mixture was washed with asaturated solution of NaHCO₃ (100 mL) and brine (100 mL) and dried overNa₂SO₄. The drying agent was removed by filtration and the solventsremoved using a rotary evaporator. The residue was purified bychromatography on silica gel (120 g RediSep column, eluting with agradient of 0% through 40% ethyl acetate/hexanes over 50 min, 85 mL(min) to give the product 39 (6.68 g, 97%) as a colorless liquid.

Example 36 (R)-3-(DECANOYLOXY)TETRADECANOIC ACID (40)

Ester 39 (10.15 g, 20.77 mmol) was dissolved in acetic acid (100 mL).Zinc (15.5 g, 237 mmol) was added, and the mixture heated to reflux for4 h. The acetic acid was removed under vacuum and the residue azeotropedwith toluene to dryness. The residue was purified by chromatography onsilica gel (120 g RediSep column, eluting with a gradient of 0% through60% ethyl acetate/hexanes over 50 min, 85 ml/min) to give the product 40(7.2 g, 89%) as a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 5.23-5.19(m, 1H), 2.62-2.55 (m, 2H), 2.34-2.25 (m, 2H), 1.65-1.58 (m, 2H),1.28-1.20 (m, 32H), 0.85 (m, 6H).

Example 37 (R)-METHYL 3-HYDROXYTETRADECANOATE (39)

A slurry of methyl 3-oxotetradecanoate (41, 5.27 g, 20.6 mmol) inmethanol (30 mL) in a 300 mL high pressure reactor glass sleeve wassparged with N₂ for 10 minutes.Dichloro-R-2,2′-bis(diphenylphosphino)-1,1′-binaphthylruthenium (142 mg,1.1 mmol) was added and the mixture was placed in a Parr 5500 seriescompact reactor. The reactor was charged with H₂ (60 psi) and ventedthree times. The reactor was then charged with a final portion of H₂ (60psi) stirred (600 rpm) and heated to 50° C. for 20 h. The reactor wasthen cooled to room temperature and the mixture concentrated in vacuo.The resulting residue was purified by silica gel chromatography, elutingwith a gradient of 0% through 50% ethyl acetate/hexanes to provide 42(3.97 g, 74%) as an off-white solid. ¹H NMR (CDCl₃) δ 4.00-3.98 (m, 1H),3.71 (s, 3H), 2.82 (d, J=6.5 Hz, 1H), 2.62-2.30 (m, 2H), 1.54-1.39 (m,3H), 1.27 (br s, 17H), (m, 20H), 0.86 (t, J=7.0 Hz, 3H).

Example 38 (R)-3-HYDROXYTETRADECANOIC ACID (36)

Lithium hydroxide monohydrate (1.98 g, 47.2 mmol) was added to asolution of (R)-methyl 3-hydroxytetradecanoate (42, 8.17 g, 31.5 mmol)in THF (66 mL) and water (66 mL) and stirred at room temperature for 2h. The mixture was then diluted with diethyl ether (1 L) and the pHadjusted to −3 with a solution of hydrochloric acid (1 N). The solutionwas then extracted with diethyl ether (200 mL), and the organicfractions were combined and dried over Na₂SO₄. Na₂SO₄ was removed byfiltration and the filtrate was concentrated in vacuo to provide(R)-3-hydroxytetradecanoic acid (36, 7.59 g, 98%) as an off-white solid.¹H NMR (CDCl₃) δ 3.99-3.94 (m, 1H), 2.45-2.39 (m, 2H), 1.47 (br s, 3H),1.29 (br s, 17H), 0.89 (t, J=7.0 Hz, 3H).

Example 39 (R)-2-OXO-2-PHENYLETHYL-3-TETRADECANOYLOXYTETRADECANOATE (46)

Myristoyl chloride (45, 8.83 g, 35.8 mmol) was added to a solution of(R)-2-oxo-2-phenylethyl 3-hydroxytetradecanoate (37, prepared accordingto Example 34, 10.8 g, 29.8 mmol) in pyridine (40 mL). The reactionmixture was stirred at room temperature for 14 h. The mixture was thenconcentrated in vacuo, and the residual pyridine removed by dissolvingthe residue in toluene (100 mL) and concentrating in vacuo. Theresulting residue was purified by silica gel chromatography, elutingwith a gradient of 0% through 20% ethyl acetate/hexanes, to provide 46(16.31 g, 83%) as a colorless oil. ¹H NMR (CDCl₃) δ 7.90 (m, 2H),7.64-7.57 (m, 1H), 7.50-7.45 (m, 2H), 5.33 (s, 2H), 5.31-5.27 (m, 1H),2.80-2.70 (m, 2H), 2.33-2.26 (t, J=4.5 Hz, 2H), 1.65-1.58 (m, 2H),1.31-1.21 (m, 40H), 0.85 (t, J=10.0 Hz, 6H).

Example 40 (R)-3-(TETRADECANOYLOXY)TETRADECANOIC ACID (47)

Zinc dust (24.42 g, 373.3 mmol) was added to a solution of 46 (16.28 g,28.42 mmol) in acetic acid (150 mL). The mixture was then heated toreflux (115° C.) for 3 h. The mixture was then concentrated in vacuo,and the residual pyridine removed by dissolving the residue in toluene(100 mL) and concentrating in vacuo. The resulting residue was by silicagel chromatography, eluting with a gradient of 0% through 30% ethylacetate/hexanes to provide (R)-benzyl 3-(tetradecanoyloxy)tetradecanoicacid (47, 11.14 g, 86% yield) as a colorless oil. ¹H NMR (CDCl₃) δ5.29-5.18 (m, 1H), 2.62-2.55 (m, 2H), 2.34-2.25 (m, 2H), 1.65-1.58 (m,3H), 1.28-1.20 (m, 40H), 0.85 (m, 6H).

Example 41TERT-BUTYLDIMETHYLSILYL-2-AZIDO-4-O-BENZYL-2-DEOXY-β-D-GLUCOPYRANOSIDE(47)

Compound 4 (prepared according to Example 3, 1.32 g, 3.36 mmol) wasdissolved in a solution of BH₃ (1 M) in THF (18.1 mL, 18.1 mmol). Afterthe mixture was stirred at 0° C. for 5 min, dibutylboron triflate (1 Min DCM, 3.62 mL, 3.62 mmol) was added dropwise, and the reaction mixturewas stirred at 0° C. for another 1 h. Subsequently, triethylamine (0.5mL) and methanol (˜0.5 mL) were added until the evolution of H₂ gas hadceased. The solvents were evaporated in vacuo, and the residue wasco-evaporated with methanol (3×50 mL). The residue was purified bysilica gel column chromatography (hexanes/ethyl acetate, 8:1 (v/v)) togive 14 (0.67 g, 49%) as a colorless oil. R₁=0.40 (hexanes/ethylacetate, 3:1 (v/v)). ¹H NMR (500 MHz, CDCl₃) δ 7.32-7.31 (m, 5H), 4.81(d, J=11.4 Hz, 1H), 4.70 (d, J=11.4 Hz, 1H), 4.55 (d, J=7.5 Hz, 1H),3.84 (m, 1H), 3.70 (dd, 1H, J=12.0, 1.5 Hz, 1H), 3.49-3.43 (m, 2H), 3.33(br s, 1H), 3.22-3.17 (m, 1H), 0.92 (s, 9H), 0.14 (s, 6H).

Example 42 Induction of Th1-Type Immune Response In Vivo

This example demonstrates in vivo Th1-type immunostimulant activity foran illustrative GLA compound of the invention having the followingstructure (IX):

Compound IX was used in a vaccine containing a Mycobacteriumtuberculosis antigenic polypeptide referred to as ID83. Standardimmunological methodologies and reagents were employed (CurrentProtocols in Immunology, Coligan et al. (Eds.) 2006 John Wiley & Sons,NY). Mice (four C57BL/6 animals per group) were immunized three times atthree-week intervals with ID83 antigen (8 μg per animal for eachimmunization) in water, ID83 antigen (8 μg per animal for eachimmunization) formulated in a stable emulsion vehicle, or ID83 antigen(8 μg per animal for each immunization) formulated in a stable emulsioncontaining (i) GLA-SE (10 μg per animal for each immunization), or (ii)Compound IX (10 μg per animal for each immunization).

One week after each injection, mice were bled to evaluateantigen-specific antibody (IgG1 and IgG2c) responses. Three weeks afterthe last immunization mice were sacrificed and spleens collected toanalyze T cell-dependent IFN-γ cytokine responses to in vitro antigenstimulation by ELISPOT according to published methods (Id.). IFN-γcytokine responses have been associated with a TH1 protective phenotypeagainst M. tuberculosis infection.

FIG. 1 shows ELISPOT data of anti-ID83 IFN-γ cytokine production inducedin mice three weeks after the third immunization using ID83 antigen andID83 component antigens (Rv2608, Rv1813 and Rv3620) formulated with astable emulsion (SE) of 10 μg Compound IX, compared to ID83 formulatedin GLA-SE, SE or water. Means and SEM of IFN-γ secreting cells permillion of splenocytes in each group are shown. “GLA-SE”, as used in theExamples herein refers to a stable emulsion of a compound as describedin co-owned U.S. Patent Publication No. 20080131466, wherein R¹, R³, R⁵and R⁶ are C₁₁ linear alkyl; and R² and R⁴ are C₁₃ linear alkyl.

All animals responded equivalently to ConA, a potent cell activator andmitogen. ID83+Compound IX vaccination induced robust ID83antigen-specific cytokine responses, while little or no such responseswere observed in the ID83+water or ID83+SE control groups. Similarlevels of IFN-γ secreting cells were elicited in splenocytes purifiedfrom mice immunized with ID83+Compound IX or ID83+GLA-SE uponrestimulation with the ID83 component antigens, Rv2608, Rv1813 andRv3620.

In conclusion, Compound IX in a stable oil formulation with M.tuberculosis vaccine antigen candidate ID83 induced predominantlyantigen-specific immune responses of the cellular type (T cell)associated with the protective TH1 phenotype.

Example 43 Induction of Th1- and Th2-Type Immune Responses In Vivo

This example demonstrates in vivo Th1- and Th2-type immunostimulantactivity of Compound IX in a vaccine containing a Mycobacteriumtuberculosis antigen referred to as ID83. Standard immunologicalmethodologies and reagents were employed (Current Protocols inImmunology, Coligan et al. (Eds.) 2006 John Wiley & Sons, NY).

Mice (four C57BL/6 animals per group) were immunized three times atthree-week intervals with the ID83 antigen (8 μg per animal for eachimmunization) used alone or formulated in a stable emulsion containingCompound IX (10 μg per animal for each immunization). Sera werecollected by bleeding animals one week after each immunization, andserum levels of IgG1 and IgG2c antibodies specific for ID83 wereexamined by ELISA according to published methods (Id.) Predominance ofeither IgG1 or IgG2c antibody isotype is associated with TH2 or TH1responses, respectively. It has been demonstrated that a TH1 response isnecessary for protection against Mycobacterium tuberculosis infection.

As shown in FIG. 2, vaccination with ID83 in water induced predominantlyantigen-specific IgG1 antibody. In contrast, ID83+SE, ID83 CompoundIX-SE or ID83 GLA-SE vaccination induced higher IgG2c antibody titers,and converted the phenotype to a mixed IgG1:IgG2c antigen-specificantibody response.

Example 44 Induction of TLR4-Dependent Immunostimulation in Human Cells

This example demonstrates the immunostimulatory activity of Compound IXin human cells. Compound IX was tested in vitro using HEK 293 cells(InvivoGen) with expression vectors encoding 1) TLR4, MD-2, and CD14, or2) TLR2 and TLR6 to define compound activity and dependence on TLR4, andto exclude activation of TLR2. These HEK 293 cell lines were furtherstably transfected with the NF-kB reporter vector pNifty-2 such thatalkaline phosphatase is secreted into the growth media upon activationof the TLR signaling pathway. Transfected cell lines were plated at5×10⁴ cells per well in a 96-well plates and stimulated for 16-24 hourscultured in medium containing serial dilutions of Compound IX and otheradjuvants. Secreted alkaline phosphatase activity was measured in theculture media using QUANTIBlue® assay (InvivoGen). The data was measuredas enhancement of NF-kB above the PBS negative control. Using thisassay, Compound IX showed greater than two-fold enhancement of NF-kB atconcentrations as low as 0.1 μg/ml (FIG. 3). The results of theseexperiments demonstrated clear TLR4 agonist activity for Compound IXthat did not appear to be associated with induction of TLR2. Compound IXwas designed based on structural considerations of the reported atomicstructure of MD2 and TLR4. As such, the fact that it binds and elicits aprofile that is similar to that of a commercially approved TLR4 agonist(MPL®) is a surprising and unexpected result. More specifically, theprofile for Compound IX advantageously plateaus rapidly asconcentrations are increased, before one would expect the cytokinelevels to rise to a point where negative side effects may exertthemselves. Thus, it is expected that Compound IX and other illustrativecompounds of the invention can be safely administered over a broad rangeof concentrations, which is highly desirable in the context ofreproducibility of clinical outcomes among patients and for the safetyin ranging a dose for adults and children. In this respect, the lowercytokine activity for Compound IX is a surprising and desirable resultthat will further facilitate its safe use in clinical formulations.

Example 45 Induction of Immunostimulatory Cytokines in Human Blood Cells

In this example, human whole blood cells were stimulated with CompoundIX and ELISA assays were performed to detect the induction ofimmunostimulatory cytokines. Serial dilutions (1:5) of Compound IX andother adjuvants were performed with phosphate buffered saline in a 96well plate for a total of 7 dilutions. 100 μl of freshly drawn humanblood from two different donors were mixed and incubated with 100 μl ofadjuvant dilutions. Following a 20 hour incubation, plates werecentrifuged and supernatants (˜70 μl) were collected, avoiding red bloodcells, and stored at −20° C. prior performing MIP-1-α and TNF-α ELISAsusing standard biochemical procedures. The results of these experimentsfurther confirmed that Compound IX has immunostimulatory activity inprimary human blood cells (FIG. 4). Additionally, these primary donorresults mimicked the results seen in human cell lines and extend theseimportant findings in relation to the possible dose ranges and safetyprofiles for this compound.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-16. (canceled)
 17. A composition comprising a compound of thefollowing formula, or a pharmaceutically acceptable salt thereof:

and an antigen, wherein the antigen is derived from or isimmunologically cross-reactive with an infectious pathogen; an epitope,biomolecule, cell or tissue associated with cancer; or an epitope,biomolecule, cell, or tissue associated with an autoimmune disease. 18.The composition of claim 17, wherein the antigen comprises a protein, aprotein fragment, a fusion protein, a whole virus, or a virus-likeparticle.
 19. The composition of claim 17, wherein the antigen is arecombinant expression construct encoding a polypeptide.
 20. Thecomposition of claim 17, wherein the infectious pathogen is a bacterium,a virus, a fungus, or a parasite.
 21. The composition of claim 17,further comprising at least one additional component selected from thegroup consisting of a Toll-like receptor (TLR) agonist, animmunostimulatory complex (ISCOM), and a co-adjuvant.
 22. Thecomposition of claim 21, wherein the co-adjuvant is selected from thegroup consisting of saponin, saponin mimetic, alum, plant alkaloid,detergent, cytokine, imidazoquinoline immune response modifier, anddouble stem loop immune modifier (dSLIM).
 23. The composition of claim17, further comprising an oil-in-water emulsion, wherein the oildroplets have a diameter of less than 1 micron.
 24. A compositioncomprising a compound of the following formula, or a pharmaceuticallyacceptable salt thereof:

and one or more components selected from the group consisting of aToll-like receptor (TLR) agonist, an immunostimulatory complex (ISCOM),and a co-adjuvant.
 25. The composition of claim 24, wherein theco-adjuvant is selected from the group consisting of a saponin, saponinmimetic, alum, plant alkaloid, detergent, cytokine, imidazoquinolineimmune response modifier, and double stem loop immune modifier (dSLIM).26. The composition of claim 24, further comprising an antigen, whereinthe antigen is derived from or is immunologically cross-reactive with aninfectious pathogen; an epitope, biomolecule, cell or tissue associatedwith cancer; or an epitope, biomolecule, cell, or tissue associated withan autoimmune disease.
 27. The composition of claim 26, wherein theinfectious pathogen is a bacterium, a virus, a fungus, or a parasite.28. The composition of claim 26, wherein the antigen comprises aprotein, a protein fragment, a fusion protein, a whole virus, or avirus-like particle.
 29. The composition of claim 26, wherein theantigen is a recombinant expression construct encoding a polypeptide.30. The composition of claim 24, wherein the composition does notcontain an antigen.
 31. The composition of claim 24, further comprisingan oil-in-water emulsion, wherein the oil droplets have a diameter ofless than 1 micron.
 32. A method of eliciting or enhancing an immuneresponse in a subject afflicted with a viral infection, bacterialinfection, fungal infection, or parasitic infection, comprisingadministering to the subject a composition comprising a compound havingthe following formula, or a pharmaceutically acceptable salt thereof:


33. The method of claim 32, wherein the composition further comprisesone or more components selected from the group consisting of a Toll-likereceptor (TLR) agonist, an immunostimulatory complex (ISCOM), and aco-adjuvant.
 34. The method of claim 32, wherein the composition furthercomprises an antigen, wherein the antigen is derived from or isimmunologically cross-reactive with a virus, a bacterium, a fungus, orparasite.
 35. The method of claim 34, wherein the antigen comprises aprotein, a protein fragment, a fusion protein, a whole virus, or avirus-like particle.
 36. The method of claim 34, wherein the antigen isa recombinant expression construct encoding a polypeptide.
 37. Themethod of claim 32, wherein the composition does not contain an antigen.38. The method of claim 33, wherein the co-adjuvant is selected from thegroup consisting of saponin, saponin mimetic, alum, plant alkaloid,detergent, cytokine, imidazoquinoline immune response modifier, anddouble stem loop immune modifier (dSLIM).
 39. The method of claim 32,wherein the composition comprises an oil-in-water emulsion, wherein theoil droplets have a diameter of less than 1 micron.
 40. A method ofeliciting or enhancing an immune response in a subject afflicted with anautoimmune disease, viral infection, bacterial infection, fungalinfection, parasitic infection, or cancer, comprising administering tothe subject a composition comprising a compound having the followingformula, or a pharmaceutically acceptable salt thereof:

and an antigen.
 41. The method of claim 40, wherein the antigen isderived from or is immunologically cross-reactive with an infectiouspathogen; an epitope, biomolecule, cell or tissue associated withcancer; or an epitope, biomolecule, cell, or tissue associated with anautoimmune disease.
 42. The method of claim 40, wherein the antigencomprises a protein, a protein fragment, a fusion protein, a wholevirus, or a virus-like particle.
 43. The method of claim 40, wherein theantigen is a recombinant expression construct encoding a polypeptide.44. The method of claim 41, wherein the infectious pathogen is abacterium, a virus, a fungus, or a parasite.
 45. The method of claim 40,wherein the composition further comprises at least one additionalcomponent selected from the group consisting of a Toll-like receptor(TLR) agonist, an immunostimulatory complex (ISCOM), and a co-adjuvant.46. The method of claim 45, wherein the co-adjuvant is selected from thegroup consisting of saponin, saponin mimetic, alum, plant alkaloid,detergent, cytokine, imidazoquinoline immune response modifier, anddouble stem loop immune modifier (dSLIM).
 47. The method of claim 45,wherein the composition comprises an oil-in-water emulsion, wherein theoil droplets have a diameter of less than 1 micron.